Antibodies to myostatin

ABSTRACT

The present invention relates to antibodies including human antibodies and antigen-binding portions thereof that bind to myostatin, and that function to inhibit myostatin. The invention also relates to human anti-myostatin antibodies and antigen-binding portions thereof. The invention also relates to antibodies that are chimeric, bispecific, derivatized, single chain antibodies or portions of fusion proteins. The invention also relates to isolated heavy and light chain immunoglobulins derived from human anti-myostatin antibodies and nucleic acid molecules encoding such immunoglobulins. The present invention also relates to methods of making human anti-myostatin antibodies, compositions comprising these antibodies and methods of using the antibodies and compositions for diagnosis and treatment. The invention also provides gene therapy methods using nucleic acid molecules encoding the heavy and/or light immunoglobulin molecules that comprise the human anti-myostatin antibodies. The invention also relates to transgenic animals or plants comprising nucleic acid molecules of the present invention.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 11/410,886filed Apr. 24, 2006, which claims the benefit of the filing date of U.S.Provisional application No. 60/674,933, filed Apr. 25, 2005. Theabove-referenced applications are herein incorporated by reference intheir entirety.

BACKGROUND OF THE INVENTION

A growing body of evidence indicates that myostatin (mstn, Growth andDifferentiation Factor-8, or GDF-8) negatively regulates skeletal musclegrowth. For example, a myostatin null mutation in a child has beenassociated with dramatic muscle hypertrophy without any obviousabnormalities (Schuelke et al. (2004) Myostatin Mutation Associated withGross Muscle Hypertrophy in a Child. New Engl. J. Med. 350:2682-8). Anegative correlation between muscle myostatin protein levels andskeletal muscle mass has also been demonstrated (Schulte, J. N. andYarasheski, K. E. (2001). Effects of resistance training on the rate ofmuscle protein synthesis in frail elderly people. Int. J. Sport Nutr.Exerc. Metab. 11 Suppl:S111-820; Walker K S et al. (2004) Resistancetraining alters plasma myostatin but not IGF-1 in healthy men. Med SciSports Exerc. 36(5):787-93.). For example, there is an increasedexpression of muscle myostatin levels with age and increased myostatinexpression has also been shown to contribute to muscle wasting inHIV-infected patients (Gonzalez-cadavid et al. (1998) Organization ofthe human myostatin gene and expression in healthy men and HIV-infectedmen with muscle wasting. PNAS 95:14938-4321). In addition, elevatedmyostatin levels are found in elderly populations. (Yarasheski K E etal., (2002) Serum myostatin-immunoreactive protein is increased in 60-92year old women and men with muscle wasting. J. Nutr. Health Aging.6(5):343-8). Myostatin also influences bone mass as myostatin-deficientmice have increased bone mineral density (Hamrick et al., (2003) BoneArchitecture and Disc Degeneration in the Lumbar Spine of Mice LackingGDF-8 (Myostatin). J. Orthopaedic Res. 21: 1025-1032 (and referencestherein).

Antibodies to circulating myostatin have been shown to cause increasedmuscle mass and improved glucose homeostasis in murine models of type 2diabetes mellitus. Inhibition of myostatin by ip injection of aneutralizing antibody increases skeletal muscle mass, lowers fastingblood glucose and improves glucose sensitivity in obese diabetic mice(Li X. et al. (2002) Inhibition of myostatin increases muscle mass andimproves glucose sensitivity in obese, diabetic mice. Poster #224, inKeystone Symposia: Diabetes Mellitus: Molecular Mechanisms, Genetics andNew Therapies). In addition, A^(y)/a mice are known to develop insulinresistance and are used as a model for type 2 diabetes. When A^(y)/amice are made devoid of myostatin by deletion of the myostatin locus,they have normal fed glucose and insulin levels, and dramatically lowerglucose levels following an exogenous glucose load relative to normalA^(y)/amice (McPherron et al. (2002) J. Clin. Invest. 109:595-601).

Considering the detrimental muscle, bone and metabolic defectsassociated with myostatin, there is an urgent need for antibodies astherapeutics that are specific for myostatin and which prevent or treatconditions by reducing myostatin activity, as well as antibodies asdiagnostics to identify individuals that are in need of treatment toreduce myostatin activity.

SUMMARY OF THE INVENTION

The invention provides anti-myostatin antibodies, nucleic acids encodingthem, vectors and host cells for producing the antibodies, compositionsand kits comprising the antibodies and methods of making and using theantibodies. Various embodiments of the invention described in thefollowing numbered paragraphs include, but are not limited to those.

-   -   1. A human, chimeric or humanized monoclonal antibody or an        antigen-binding portion thereof that specifically binds to        myostatin.    -   2. The monoclonal antibody or antigen-binding portion according        to paragraph 1 wherein the myostatin is human myostatin.    -   3. A monoclonal antibody or antigen-binding portion according to        paragraph 1, wherein said antibody or portion selectively binds        myostatin over GDF11 by at least 50-fold.    -   4. A monoclonal antibody or antigen-binding portion according to        paragraph 3, wherein said antibody or portion inhibits myostatin        binding to an activin type I or type II receptor.    -   5. A monoclonal antibody or antigen-binding portion according to        paragraph 1, wherein the antibody or portion thereof has at        least one property selected from the group consisting of:        -   (a) competes for binding to myostatin with an antibody            selected from the group consisting of 1_(—)116_(—)1;            1_(—)136_(—)3; 1_(—)257_(—)1; 1_(—)46_(—)1; 2_(—)112_(—);            1_(—)314_(—)1; 1_(—)66_(—)1; 2_(—)43_(—)1; 2_(—)177_(—)1;            1_(—)132_(—)1; 1_(—)268_(—)1; 2_(—)112_K;            1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I;            1_(—)314_(—)1H-T92A; 1_(—)46_(—)1H-L81M;            2_(—)112_(—)1H-I12V; 2_(—)112_(—)1L-F58I;            2_(—)112_(—)1L-I85V; 2_(—)112_(—)1H-L81M, L-F58I;            2_(—)112_(—)1H-L81M, L-I85V; and 2_(—)112_(—)1H-L81M,            L-F58I, I85V;        -   (b) binds to the same epitope of myostatin as an antibody            selected from the group consisting of 1_(—)116_(—)1;            1_(—)136_(—)3; 1_(—)257_(—)1; 1_(—)46_(—)1; 2_(—)112_(—);            1_(—)314_(—)1; 1_(—)66_(—)1; 2_(—)43_(—)1; 2_(—)177_(—)1;            1_(—)132_(—)1; 1_(—)268_(—)1; 2_(—)112_K;            1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I;            1_(—)314_(—)1H-T92A; 1_(—)46_(—)1H-L81M;            2_(—)112_(—)1H-I12V; 2_(—)112_(—)1L-F58I;            2_(—)112_(—)1L-I85V; 2_(—)112_(—)1H-L81M, L-F58I;            2_(—)112_(—)1H-L81M, L-I85V; and 2_(—)112_(—)1H-L81M,            L-F58I, I85V;        -   (c) binds to myostatin with substantially the same K_(D) as            an antibody selected from the group consisting of            1_(—)116_(—)1; 1_(—)136_(—)3; 1_(—)257_(—)1; 1_(—)46_(—)1;            2_(—)112_(—)1; 1_(—)314_(—)1; 1_(—)66_(—)1; 2_(—)43_(—)1;            2_(—)177_(—)1; 1_(—)132_(—)1; 1_(—)268_(—)1; 2_(—)112_K;            1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I;            1_(—)314_(—)1H-T92A; 1_(—)46_(—)1H-L81M;            2_(—)112_(—)1H-I12V; 2_(—)112_(—)1L-F58I;            2_(—)112_(—)1L-I85V; 2_(—)112_(—)1H-L81M, L-F58I;            2_(—)112_(—)1H-L81M, L-I85V; and 2_(—)112_(—)1H-L81M,            L-F58I, I85V; and        -   (d) binds to myostatin with substantially the same off rate            as an antibody selected from the group consisting of            1_(—)116_(—)1; 1_(—)136_(—)3; 1_(—)257_(—)1; 1_(—)46_(—)1;            2_(—)112_(—)1; 1_(—)314_(—)1; 1_(—)66_(—)1; 2_(—)43_(—)1;            2_(—)177_(—)1; 1_(—)132_(—)1; 1_(—)268_(—)1; 2_(—)112_K;            1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I;            1_(—)314_(—)1H-T92A; 1_(—)46_(—)1H-L81M;            2_(—)112_(—)1H-I12V; 2_(—)112_(—)1L-F58I;            2_(—)112_(—)1L-I85V; 2_(—)112_(—)1H-L81M, L-F58I;            2_(—)112_(—)1H-L81M, L-I85V; and 2_(—)112_(—)1H-L81M,            L-F58I, I85V.    -   6. A monoclonal antibody or antigen-binding portion thereof        comprising:        -   (a) a CDR set, CDR1, CDR2, and CDR3, that sequentially            together are at least 90% identical in amino acid sequence            to heavy chain CDRs, CDR1, CDR2, and CDR3, sequentially            together, that are included in the amino acid sequence set            forth in any one of SEQ ID NOs: 45, 49, 53, 57, 61, 65, 69,            73, 77, 81, 85 and 118;        -   (b) a CDR set, CDR1, CDR2, and CDR3, that sequentially            together are at least 90% identical in amino acid sequence            to light chain CDRs, CDR1, CDR2, and CDR3, sequentially            together, that are included in the amino acid sequence set            forth in any one of SEQ ID NOs: 47, 51, 55, 59, 63, 67, 71,            75, 83, 87 and 120; or        -   (c) a first CDR set of (a) and a second CDR set of (b).    -   7. A monoclonal antibody or antigen-binding portion thereof        according to paragraph 1, wherein said antibody comprises heavy        chain CDRs CDR1, CDR2, and CDR3, that sequentially together are        at least 90% identical in amino acid sequence to heavy chain        CDRs, CDR1, CDR2, and CDR3, sequentially together, that are        included in the amino acid sequence set forth in any one of SEQ        ID NOs: 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85 and 118.    -   8. A monoclonal antibody or antigen-binding portion thereof        according to paragraph 1, wherein said antibody comprises light        chain CDRs CDR1, CDR2, and CDR3, that sequentially together are        at least 90% identical in amino acid sequence to light chain        CDRs, CDR1, CDR2, and CDR3, sequentially together, that are        included in the amino acid sequence set forth in any one of SEQ        ID NOs: 47, 51, 55, 59, 63, 67, 71, 75, 83, 87 and 120.    -   9. A monoclonal antibody or antigen-binding portion according to        paragraph 1, wherein said antibody or antigen-binding portion        comprises the heavy chain CDR1, CDR2 and CDR3 sequences found in        any one of SEQ ID NOs: 45, 49, 53, 57, 61, 65, 69, 73, 77, 81,        85 or 118.    -   10. A monoclonal antibody or antigen-binding portion according        to paragraph 1, wherein said antibody or antigen-binding portion        comprises the light chain CDR1, CDR2 and CDR3 sequences found in        any one of SEQ ID NOs: 47, 51, 55, 59, 63, 67, 71, 75, 79, 83,        87 or 120.    -   11. A monoclonal antibody or antigen-binding portion according        to paragraph 1, wherein said antibody or portion comprises a        heavy chain that utilizes a human V_(H) 1-02 gene, a human V_(H)        3-21 gene or a human V_(H) 3-23 gene.    -   12. A monoclonal antibody or an antigen-binding portion        according to paragraph 1, wherein said antibody or portion        comprises a light chain that utilizes a human V_(K) L2 gene, a        human V_(K) A3 gene or a human V_(K) A30 gene.    -   13. A monoclonal antibody according to paragraph 1 comprising a        V_(H) domain at least 90% identical in amino acid sequence to        the V_(H) domain in any one of SEQ ID NOs: 2, 6, 10, 14, 18, 22,        26, 30, 34, 38, 42 or 115.    -   14. A monoclonal antibody according to paragraph 1 comprising a        V_(L) domain at least 90% identical in amino acid sequence to        the V_(L) domain in any one of SEQ ID NOs:4, 8, 12, 16, 20, 24,        28, 32, 36, 40, 44 or 117.    -   15. A monoclonal antibody or an antigen-binding portion        according to paragraph 1 that specifically binds myostatin,        wherein:        -   (a) the heavy chain comprises the heavy chain CDR1, CDR2 and            CDR3 amino acid sequences of an antibody selected from the            group consisting of: 1_(—)116_(—)1; 1_(—)136_(—)3;            1_(—)257_(—)1; 1_(—)46_(—)1; 2_(—)112_(—); 1_(—)314_(—)1;            1_(—)66_(—)1; 2_(—)43_(—)1; 2_(—)177_(—)1; 1_(—)132_(—)1;            1_(—)268_(—)1; 2_(—)112_K; 1_(—)116_(—)1L-Q45K;            1_(—)257_(—)1L-L21I; 1_(—)314_(—)1H-T92A;            1_(—)46_(—)1H-L81M; 2_(—)112_(—)1H-I12V;            2_(—)112_(—)1L-F58I; 2_(—)112_(—)1L-I85V;            2_(—)112_(—)1H-L81M, L-F58I; 2_(—)112_(—)1H-L81M, L-I85V;            and 2_(—)112_(—)1H-L81M, L-F58I, I85V;        -   (b) the light chain comprises the light chain CDR1, CDR2 and            CDR3 amino acid sequences of an antibody selected from the            group consisting of 1_(—)116_(—)1; 1_(—)136_(—)3;            1_(—)257_(—)1; 1_(—)46_(—)1; 2_(—)112_(—); 1_(—)314_(—)1;            1_(—)66_(—)1; 2_(—)43_(—)1; 2_(—)177_(—)1; 1_(—)132_(—)1;            1_(—)268_(—)1; 2_(—)112_K; 1_(—)116_(—)1L-Q45K;            1_(—)257_(—)1L-L21I; 1_(—)314_(—)1H-T92A;            1_(—)46_(—)1H-L81M; 2_(—)112_(—)1H-I12V;            2_(—)112_(—)1L-F58I; 2_(—)112_(—)1L-I85V;            2_(—)112_(—)1H-L81M, L-F58I; 2_(—)112_(—)1H-L81M, L-I85V;            and 2_(—)112_(—)1H-L81M, L-F58I, I85V; or        -   (c) the antibody comprises a heavy chain of (a) and a light            chain of (b).    -   16. A monoclonal antibody according to paragraph 12 further        comprising a V_(L) domain at least 90% identical in amino acid        sequence to the variable domain in any one of SEQ ID NOs: 4, 8,        12, 16, 20, 24, 28, 32, 36, 40, 44 or 117.    -   17. A monoclonal antibody selected from the group consisting of:        -   (a) an antibody comprising the amino acid sequences set            forth in SEQ ID NO: 2 and SEQ ID NO: 4.        -   (b) an antibody comprising the amino acid sequences set            forth in SEQ ID NO: 6 and SEQ ID NO: 8;        -   (c) an antibody comprising the amino acid sequences set            forth in SEQ ID NO: 10 and SEQ ID NO: 12;        -   (d) an antibody comprising the amino acid sequences set            forth in SEQ ID NO: 14 and SEQ ID NO: 16;        -   (e) an antibody comprising the amino acid sequences set            forth in SEQ ID NO: 18 and SEQ ID NO: 20;        -   (f) an antibody comprising the amino acid sequences set            forth in SEQ ID NO: 22 and SEQ ID NO: 24;        -   (g) an antibody comprising the amino acid sequences set            forth in SEQ ID NO: 26 and SEQ ID NO: 28;        -   (h) an antibody comprising the amino acid sequences set            forth in SEQ ID NO: 30 and SEQ ID NO: 32;        -   (i) an antibody comprising the amino acid sequences set            forth in SEQ ID NO: 34 and SEQ ID NO: 36;        -   (j) an antibody comprising the amino acid sequences set            forth in SEQ ID NO: 38 and SEQ ID NO: 40;        -   (k) an antibody comprising the amino acid sequences set            forth in SEQ ID NO: 42 and SEQ ID NO: 44; and        -   (l) an antibody comprising the amino acid sequences set            forth in SEQ ID NO: 115 and SEQ ID NO: 117.    -   18. A monoclonal antibody or antigen-binding portion thereof        selected from the group consisting of:        -   (a) an antibody or antigen-binding portion comprising the            variable domain amino acid sequences set forth in SEQ ID NO:            2 and SEQ ID NO: 4.        -   (b) an antibody or antigen-binding portion comprising the            variable domain amino acid sequences set forth in SEQ ID NO:            6 and SEQ ID NO: 8;        -   (c) an antibody or antigen-binding portion comprising the            variable domain amino acid sequences set forth in SEQ ID NO:            10 and SEQ ID NO: 12;        -   (d) an antibody or antigen-binding portion comprising the            variable domain amino acid sequences set forth in SEQ ID NO:            14 and SEQ ID NO: 16;        -   (e) an antibody or antigen-binding portion comprising the            variable domain amino acid sequences set forth in SEQ ID NO:            18 and SEQ ID NO: 20;        -   (f) an antibody or antigen-binding portion comprising the            variable domain amino acid sequences set forth in SEQ ID NO:            22 and SEQ ID NO: 24;        -   (g) an antibody or antigen-binding portion comprising the            variable domain amino acid sequences set forth in SEQ ID NO:            26 and SEQ ID NO: 28;        -   (h) an antibody or antigen-binding portion comprising the            variable domain amino acid sequences set forth in SEQ ID NO:            30 and SEQ ID NO: 32;        -   (i) an antibody or antigen-binding portion comprising the            variable domain amino acid sequences set forth in SEQ ID NO:            34 and SEQ ID NO: 36;        -   (j) an antibody or antigen-binding portion comprising the            variable domain amino acid sequences set forth in SEQ ID NO:            38 and SEQ ID NO: 40;        -   (k) an antibody or antigen-binding portion comprising the            variable domain amino acid sequences set forth in SEQ ID NO:            42 and SEQ ID NO: 44; and        -   (l) an antibody or antigen-binding portion comprising the            variable domain amino acid sequences set forth in SEQ ID NO:            115 and SEQ ID NO: 117.    -   19. A monoclonal antibody or antigen-binding portion thereof        comprising a first CDR sequence set comprising a first CDR1,        first CDR2 and first CDR3 and a second CDR sequence set        comprising a second CDR1, second CDR2 and second CDR3, wherein        said first CDR set and said second CDR set each sequentially        together have at least 90% identity to the CDR1, CDR2 and CDR3        sequences, sequentially together, of:        -   (a) SEQ ID NO: 2 and SEQ ID NO: 4, respectively;        -   (b) SEQ ID NO: 6 and SEQ ID NO: 8, respectively;        -   (c) SEQ ID NO:10 and SEQ ID NO:12, respectively;        -   (d) SEQ ID NO:14 and SEQ ID NO:16, respectively;        -   (e) SEQ ID NO:18 and SEQ ID NO:20, respectively;        -   (f) SEQ ID NO:22 and SEQ ID NO:24, respectively;        -   (g) SEQ ID NO:26 and SEQ ID NO:28, respectively;        -   (h) SEQ ID NO:30 and SEQ ID NO:32, respectively;        -   (i) SEQ ID NO:34 and SEQ ID NO:36, respectively;        -   (j) SEQ ID NO:38 and SEQ ID NO:40, respectively;        -   (k) SEQ ID NO:42 and SEQ ID NO:44, respectively; and        -   (l) SEQ ID NO:115 and SEQ ID NO:117, respectively.    -   20. A monoclonal antibody or antigen-binding portion thereof        comprising a first CDR sequence set comprising a first CDR1,        first CDR2 and first CDR3 and a second CDR sequence set        comprising a second CDR1, second CDR2 and second CDR3, wherein        said first CDR set and said second CDR set are each the CDR1,        CDR2 and CDR3 sequences, sequentially together, of        -   a) SEQ ID NO: 2 and SEQ ID NO: 4, respectively;        -   (b) SEQ ID NO: 6 and SEQ ID NO: 8, respectively;        -   (c) SEQ ID NO:10 and SEQ ID NO:12, respectively;        -   (d) SEQ ID NO:14 and SEQ ID NO:16, respectively;        -   (e) SEQ ID NO:18 and SEQ ID NO:20, respectively;        -   (f) SEQ ID NO:22 and SEQ ID NO:24, respectively;        -   (g) SEQ ID NO:26 and SEQ ID NO:28, respectively;        -   (h) SEQ ID NO:30 and SEQ ID NO:32, respectively;        -   (i) SEQ ID NO:34 and SEQ ID NO:36, respectively;        -   (k) SEQ ID NO:38 and SEQ ID NO:40, respectively;        -   (l) SEQ ID NO:42 and SEQ ID NO:44, respectively; and        -   (m) SEQ ID NO:115 and SEQ ID NO:117, respectively.    -   21. A monoclonal antibody that specifically binds myostatin        comprising the heavy chain amino acid sequence set forth in SEQ        ID NO:115 and the light chain amino acid sequence set forth in        SEQ ID NO:117.    -   22. A monoclonal antibody or an antigen-binding portion thereof        comprising the variable regions contained in SEQ ID NO:115 and        SEQ ID NO:117.    -   23. A monoclonal antibody or an antigen-binding portion thereof,        that specifically binds myostatin comprising CDRs CDR1, CDR2,        and CDR3 contained in SEQ ID NO:115 and SEQ ID NO:117.    -   24. A monoclonal antibody or an antigen-binding portion thereof        said monoclonal antibody or antigen-binding portion binds to        peptide 1 and peptide 5 portions of myostatin, wherein peptide 1        comprises the amino acid sequence of SEQ ID NO: 103 and peptide        5 comprises the amino acid sequence of SEQ ID NO: 107.    -   25. An antibody produced by a cell having ATCC Deposit        Designation Number selected from the group consisting of        PTA-6566, PTA-6567, PTA-6568, PTA-6569, PTA-6570, PTA-6571,        PTA-6572, PTA-6573, PTA-6574, PTA-6575, and PTA-6576.    -   26. A pharmaceutical composition comprising an antibody or an        antigen-binding portion according to any one of paragraphs 1 to        25 and a pharmaceutically acceptable carrier.    -   27. A pharmaceutical composition according to paragraph 26,        further comprising at least one therapeutic agent.    -   28. A method comprising the step of administering to said        subject an antibody or an antigen-binding portion according to        any one of paragraphs 1 to 25 or the pharmaceutical composition        according to paragraph 26, wherein said antibody,        antigen-binding portion or pharmaceutical composition inhibits        myostatin activity, wherein said subject is in need of        increasing muscle mass, promoting skeletal muscle development,        treating a muscle wasting disorder or enhancing skeletal muscle        growth.    -   29. An isolated cell line that produces an antibody or an        antigen-binding portion according to any one of paragraphs 1 to        25 or the heavy chain or light chain of said antibody or said        antigen-binding portion.    -   30. An isolated nucleic acid molecule comprising a nucleotide        sequence that encodes the heavy chain or an antigen-binding        portion thereof or the light chain or an antigen-binding portion        thereof of an antibody according to any one of paragraphs 1 to        25.    -   31. A vector comprising the nucleic acid molecule according to        paragraph 30 wherein the vector optionally comprises an        expression control sequence operably linked to the nucleic acid        molecule.    -   32. A host cell comprising the vector according to paragraph 31        or the nucleic acid molecule according to paragraph 30.    -   33. A method for producing an anti-myostatin antibody or an        antigen-binding portion thereof, comprising culturing the host        cell according to paragraph 32 or the cell line according to        paragraph 29 under suitable conditions and recovering said        antibody or antigen-binding portion.    -   34. A non-human transgenic organism carrying the nucleic acid        according to paragraph 30 either chromosomally or        extrachromosomally, wherein the non-human transgenic organism        expresses said nucleic acid.    -   35. A method for isolating an antibody or an antigen-binding        portion thereof that specifically binds to myostatin, comprising        the step of isolating the antibody from the non-human transgenic        organism according to paragraph 34.    -   36. A method for treating a subject in need thereof with an        antibody or an antigen-binding portion thereof that specifically        binds to myostatin comprising the steps of:        -   (a) administering to said subject an effective amount of an            isolated nucleic acid molecule according to paragraph 30;            and        -   (b) expressing said nucleic acid molecule.    -   37. A method for producing a human monoclonal antibody that        specifically binds to myostatin, comprising the steps of:        -   (a) immunizing a non-human transgenic animal that is capable            of producing human antibodies with myostatin, an immunogenic            portion of myostatin, or a cell or tissue expressing            myostatin;        -   (b) allowing the non-human transgenic animal to mount an            immune response to myostatin;        -   (c) isolating B lymphocytes from the non-human transgenic            animal; and        -   (d) isolating a monoclonal antibody that specifically binds            to myostatin from said isolated B lymphocytes.    -   38. An isolated antibody produced by the method according to        paragraph 37.    -   39. A method for inhibiting the binding of myostatin to cells        expressing an activin Type II or IIB receptor comprising        contacting the myostatin with an antibody or antigen-binding        portion according to any one of paragraphs 1 to 25, wherein said        antibody or antigen-binding portion inhibits myostatin activity.    -   40. A method for increasing myoblast proliferation comprising        contacting a composition comprising myoblasts and myostatin with        an antibody or antigen-binding portion according to any one of        paragraphs 1 to 25, wherein said antibody or antigen-binding        portion inhibits myostatin activity.    -   41. A method comprising administering to a subject an antibody        or an antigen-binding portion thereof according to any one of        paragraphs 1 to 25, wherein said antibody or antigen-binding        portion inhibits myostatin activity, wherein said subject is in        need of improving glucose homeostasis, decreasing fat mass,        increasing insulin sensitivity, improving kidney function,        decreasing fat accumulation, treating, preventing or inhibiting        a disease or condition characterized by bone loss, said disease        or condition including osteoporosis, osteopenia, osteoarthritis        and bone fractures, treating metabolic syndrome, or        counteracting muscle wasting from sustained administration of a        glucocorticoid or a steroid hormone during the time that said        subject is undergoing treatment with a glucocorticoid or a        steroid hormone.    -   42. A method for reducing myostatin activity in a subject in        need thereof comprising the step of administering to said        subject a monoclonal antibody or an antigen-binding portion        thereof according to any one of paragraphs 1 to 25, wherein said        monoclonal antibody or antigen-binding portion inhibits        myostatin activity.    -   43. A method for reversing age-related decline in muscle mass in        a subject in need thereof comprising the step of administering        to said subject an antibody or an antigen-binding portion        according to any one of paragraphs 1 to 25, wherein said        antibody or antigen-binding portion inhibits myostatin activity.    -   44. A method for increasing myoblast proliferation and        differentiation in a subject in need thereof comprising the step        of administering to said subject an antibody or antigen-binding        portion according to any one of paragraphs 1 to 25, wherein said        antibody or antigen-binding portion inhibits myostatin activity.    -   45. A method for reducing myostatin-induced activin type IIA or        IIB membrane receptor mediated cell signalling in a subject in        need thereof comprising the step of administering to said        subject an antibody or an antigen-binding portion according to        any one of paragraphs 1 to 25, wherein said antibody or        antigen-binding portion inhibits myostatin activity.    -   46. A method for decreasing myostatin-mediated activation of an        activin type I membrane receptor in a subject in need thereof        comprising the step of administering to said subject an antibody        or an antigen-binding portion according to any one of paragraphs        1 to 25, wherein said antibody or antigen-binding portion        inhibits myostatin activity.    -   47. A method for reducing myostatin-mediated phosphorylation of        one or more R-smad proteins selected from the group consisting        of: Smad 2 and Smad 3 in a subject in need thereof comprising        the step of administering to said subject an antibody or        antigen-binding portion according to any one of paragraphs 1 to        18, wherein said antibody or antigen-binding portion inhibits        myostatin activity.    -   48. A method for increasing expression of a gene selected from        the group consisting of: myoD, myf5 and myogenin, in a subject        in need thereof comprising the step of administering to said        subject an antibody or antigen-binding portion according to any        one of paragraphs 1 to 25, wherein said antibody or        antigen-binding portion inhibits myostatin activity.    -   49. A method for promoting muscle growth, weight gain or aiding        in the prevention of frailty in cattle, swine, sheep, chickens,        turkeys, horses, fish, dogs and cats in need thereof comprising        the step of administering to said subject an antibody or        antigen-binding portion according to any one of paragraphs 1 to        25, wherein said antibody or antigen-binding portion inhibits        myostatin activity.    -   50. A monoclonal antibody or antigen binding portion thereof        selected from the group consisting of:        -   (a) an antibody or antigen binding portion thereof            comprising the variable domain amino acid sequences set            forth in SEQ ID NO: 45 and SEQ ID NO:47;        -   (b) an antibody or antigen binding portion thereof            comprising the variable domain amino acid sequences set            forth in SEQ ID NO:49 and SEQ ID NO:51;        -   (c) an antibody or antigen binding portion thereof            comprising the variable domain amino acid sequences set            forth in SEQ ID NO: 53 and SEQ ID NO:55;        -   (d) an antibody or antigen binding portion thereof            comprising the variable domain amino acid sequences set            forth in SEQ ID NO:57 and SEQ ID NO:59;        -   (e) an antibody or antigen binding portion thereof            comprising the variable domain amino acid sequences set            forth in SEQ ID NO:61 and SEQ ID NO:63;        -   (f) an antibody or antigen binding portion thereof            comprising the variable domain amino acid sequences set            forth in SEQ ID NO:65 and SEQ ID NO:67;        -   (g) an antibody or antigen binding portion thereof            comprising the variable domain amino acid sequences set            forth in SEQ ID NO:69 and SEQ ID NO:71;        -   (h) an antibody or antigen binding portion thereof            comprising the variable domain amino acid sequences set            forth in SEQ ID NO:73 and SEQ ID NO:75;        -   (i) an antibody or antigen binding portion thereof            comprising the variable domain amino acid sequences set            forth in SEQ ID NO:77 and SEQ ID NO:79;        -   (j) an antibody or antigen binding portion thereof            comprising the variable domain amino acid sequences set            forth in SEQ ID NO:81 and SEQ ID NO:83; and        -   (k) an antibody or antigen binding portion thereof            comprising the variable domain amino acid sequences set            forth in SEQ ID NO:85 and SEQ ID NO:87; and        -   (l) an antibody or antigen binding portion thereof            comprising the variable domain amino acid sequences set            forth in SEQ ID NO: 118 and SEQ ID NO:120.    -   51. A method of treating metabolic syndrome in a subject in need        thereof comprising the step of administering to said subject an        antibody or antigen-binding portion according to any one of        paragraphs 1 to 25, wherein said antibody or antigen-binding        portion inhibits myostatin activity.    -   52. A mammalian host cell line comprising polynucleotides        encoding the heavy and light chains of a human, chimeric or        humanized monoclonal antibody that competes for binding to        myostatin with an antibody or an antigen-binding portion        thereof, wherein the antibody or portion thereof has at least        one property selected from the group consisting of:        -   (a) competes for binding to myostatin with an antibody            selected from the group consisting of 1_(—)116_(—)1;            1_(—)136_(—)3; 1_(—)257_(—)1; 1_(—)46_(—)1; 2_(—)112_(—);            1_(—)314_(—)1; 1_(—)66_(—)1; 2_(—)43_(—)1; 2_(—)177_(—)1;            1_(—)132_(—)1; 1_(—)268_(—)1; 2_(—)112_K;            1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I;            1_(—)314_(—)1H-T92A; 1_(—)46_(—)1H-L81M;            2_(—)112_(—)1H-I12V; 2_(—)112_(—)1L-F58I;            2_(—)112_(—)1L-I85V; 2_(—)112_(—)1H-L81M, L-F58I;            2_(—)112_(—)1H-L81M, L-I85V; and 2_(—)112_(—)1H-L81M,            L-F58I, I85V;        -   (b) binds to the same epitope of myostatin as an antibody            selected from the group consisting of 1_(—)116_(—)1;            1_(—)136_(—)3; 1_(—)257_(—)1; 1_(—)46_(—)1; 2_(—)112_(—);            1_(—)314_(—)1; 1_(—)66_(—)1; 2_(—)43_(—)1; 2_(—)177_(—)1;            1_(—)132_(—)1; 1_(—)268_(—)1; 2_(—)112_K;            1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I; 1            _(—)314_(—)1H-T92A; 1_(—)46_(—)1H-L81M; 2_(—)112_(—)1H-I12V;            2_(—)112_(—)1L-F58I; 2_(—)112_(—)1L-I85V;            2_(—)112_(—)1H-L81M, L-F58I; 2_(—)112_(—)1H-L81M, L-I85V;            and 2_(—)112_(—)1H-L81M, L-F58I, I85V;        -   (c) binds to myostatin with substantially the same K_(D) as            an antibody selected from the group consisting of            1_(—)116_(—)1; 1_(—)136_(—)3; 1_(—)257_(—)1; 1_(—)46_(—)1;            2_(—)112_(—)1; 1_(—)314_(—)1; 1_(—)66_(—)1; 2_(—)43_(—)1;            2_(—)177_(—)1; 1_(—)132_(—)1; 1_(—)268_(—)1; 2_(—)112_K;            1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I;            1_(—)314_(—)1H-T92A; 1_(—)46_(—)1H-L81M;            2_(—)112_(—)1H-I12V; 2_(—)112_(—)1L-F58I;            2_(—)112_(—)1L-I85V; 2_(—)112_(—)1H-L81M, L-F58I;            2_(—)112_(—)1H-L81M, L-I85V; and 2_(—)112_(—)1H-L81M;            L-F58I, I85V; and        -   (d) binds to myostatin with substantially the same off rate            as an antibody selected from the group consisting of            1_(—)116_(—)1; 1_(—)136_(—)3; 1_(—)257_(—)1; 1_(—)46_(—)1;            2_(—)112_(—)1; 1_(—)314_(—)1; 1_(—)66_(—)1; 2_(—)43_(—)1;            2_(—)177_(—)1; 1_(—)132_(—)1; 1_(—)268_(—)1; 2_(—)112_K;            1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I;            1_(—)314_(—)1H-T92A; 1_(—)46_(—)1H-L81M;            2_(—)112_(—)1H-I12V; 2_(—)112_(—)1L-F58I;            2_(—)112_(—)1L-I85V; 2_(—)112_(—)1H-L81M, L-F58I;            2_(—)112_(—)1H-L81M, L-I85V; and 2_(—)112_(—)1H-L81M;            L-F58I, I85V.    -   53. A mammalian host cell line comprising polynucleotides        encoding the heavy and light chains of a monoclonal antibody or        antigen-binding portion thereof that competes for binding to        myostatin with an antibody comprising:        -   (a) a heavy chain that utilizes a human V_(H) 1-02 gene, a            human V_(H) 3-21 gene or a human V_(H) 3-23 gene; and        -   (b) a light chain that utilizes a human V_(K) L2 gene, a            human V_(K) A3 gene or a human V_(K) A30 gene.    -   54. A mammalian host cell line comprising polynucleotides        encoding the heavy and light chains of a monoclonal antibody or        an antigen-binding portion of said monoclonal antibody having        the same amino acid sequence as the antibody produced by a        hybridoma cell line having ATCC Deposit Designation Number        selected from the group consisting of PTA-6566, PTA-6567,        PTA-6568, PTA-6569, PTA-6570, PTA-6571, PTA-6572, PTA-6573,        PTA-6574, PTA-6575, and PTA-6576.    -   55. A mammalian host cell line comprising polynucleotides        encoding an antibody having the same amino acid sequence as the        antibody produced by a hybridoma cell having an ATCC Deposit        Designation Number selected from the group consisting of        PTA-6566, PTA-6567, PTA-6568, PTA-6569, PTA-6570, PTA-6571,        PTA-6572, PTA-6573, PTA-6574, PTA-6575, and PTA-6576.    -   56. A method comprising expressing said human monoclonal        antibody in said mammalian host cell line of any one of        paragraphs 52-55 and recovering said human monoclonal antibody.    -   57. A hybridoma cell line selected from the group consisting of        ATCC Deposit Designation Numbers PTA-6566, PTA-6567, PTA-6568,        PTA-6569, PTA-6570, PTA-6571, PTA-6572, PTA-6573, PTA-6574,        PTA-6575, and PTA-6576    -   58. A. mammalian host cell line comprising polynucleotides        encoding the heavy and light chains of a human, chimeric or        humanized monoclonal antibody, wherein the antibody or portion        thereof has at least one property selected from the group        consisting of:        -   (a) competes for binding to myostatin with an antibody            selected from the group consisting of: 1_(—)116_(—)1;            1_(—)136_(—)3; 1_(—)257_(—)1; 1_(—)46_(—)1; 2_(—)112_(—);            1_(—)314_(—)1; 1_(—)66_(—)1; 2_(—)43_(—)1; 2_(—)177_(—)1;            1_(—)132_(—)1; 1_(—)268_(—)1; 2_(—)112_K;            1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I;            1_(—)314_(—)1H-T92A; 1_(—)46_(—)1H-L81M;            2_(—)112_(—)1H-I12V; 2_(—)112_(—)1L-F58I;            2_(—)112_(—)1L-I85V; 2_(—)112_(—)1H-L81M, L-F58I;            2_(—)112_(—)1H-L81M, L-I85V; and 2_(—)112_(—)1H-L81M,            L-F58I, I85V;        -   (b) binds to the same epitope of myostatin as an antibody            selected from the group consisting of: 1_(—)116_(—)1;            1_(—)136_(—)3; 1_(—)257_(—)1; 1_(—)46_(—)1; 2_(—)112_(—);            1_(—)314_(—)1; 1_(—)66_(—)1; 2_(—)43_(—)1; 2_(—)177_(—)1;            1_(—)132_(—)1; 1_(—)268_(—)1; 2_(—)112_K;            1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I;            1_(—)314_(—)1H-T92A; 1_(—)46_(—)1H-L81M;            2_(—)112_(—)1H-I12V; 2_(—)112_(—)1L-F58I;            2_(—)112_(—)1L-I85V; 2_(—)112_(—)1H-L81M, L-F58I;            2_(—)112_(—)1H-L81M, L-I85V; and 2_(—)112_(—)1H-L81M,            L-F58I, I85V;        -   (c) is an antibody having the same amino acid sequence as            the antibody produced by a hybridoma cell having an ATCC            Deposit Designation Number selected from the group            consisting of PTA-6566, PTA-6567, PTA-6568, PTA-6569,            PTA-6570, PTA-6571, PTA-6572, PTA-6573, PTA-6574, PTA-6575,            and PTA-6576; and        -   (d) is a hybridoma cell line selected from the group            consisting of ATCC Deposit Designation Numbers PTA-6566,            PTA-6567, PTA-6568, PTA-6569, PTA-6570, PTA-6571, PTA-6572,            PTA-6573, PTA-6574, PTA-6575, and PTA-6576.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a myostatin responsive reporter gene assay.As shown, neutralizing anti-myostatin antibodies inhibit myostatininduced luciferase activity in A204 cells. Human antibody variants1_(—)116_(—)1L-Q45K; 1_(—)257-1L-L21I; 1_(—)314_(—)1H-T92A and2_(—)112_(—)1H-I12V, L-F 58I, I85V inhibited luciferase activity to thesame extent as wild type antibodies.

FIG. 2 shows the results of an L6 Aurora beta-lactamase assay. As shown,neutralizing anti-myostatin antibodies inhibit myostatin inducedbeta-lactamase activity in L6 rat myoblasts. Human antibody variants1_(—)116_(—)1L-Q45K; 1_(—)257-1L-L21I; 1_(—)314_(—)1H-T92A and2_(—)112_(—)1H-I12V, L-F58I, I85V inhibited beta-lactamase activity tothe same extent as the wild type antibodies.

FIG. 3 shows the results of a western blot. As shown, neutralizinganti-myostatin antibodies of the invention inhibit myostatin inducedsmad2 phosphorylation in HepG2 cells by western blot.

FIG. 4 shows the results of myf5 mRNA expression assays. (A)Neutralizing anti-myostatin antibodies 1_(—)268_(—)1, 2_(—)177_(—)1, and2_(—)112_(—)1 rescued myf5 gene expression inhibited by myostatin inC2C12 mouse myoblasts, whereas a non-neutralizing antibody 1_(—)159_(—)1could not. Myf5 mRNA level was detected by TAQMAN® PCR. (B)1_(—)116_(—)1 rescued myf5 gene expression in a dose-dependent manner.

FIG. 5 shows the results of C2C12 muscle cell differentiation assays.

(A) Neutralizing anti-myostatin antibodies 2_(—)43_(—)1, 1_(—)314_(—)1,and 1_(—)257_(—)1 rescued myostatin-blocked muscle differentiation inC2C12 mouse muscle cells. Embryonic myosin heavy chain (MHC) proteinlevel was used a marker to measure C2C12 differentiation. (B) Antibodies2_(—)112_(—)1, 1_(—)46_(—)1, and 1_(—)116_(—)1 rescued myostatin-blockedC2C12 muscle differentiation.

FIG. 6 (A) illustrates peptides generated from mature GDF8 (SEQ ID NO:89) to test anti-myostatin antibody binding. Amino acids in lower caseletters are the amino acids that are different from GDF11 (B) Summary ofpeptide binding. As shown in the Figure, some antibodies do not bind toany of the peptides. Some antibodies bind to non-contiguous peptides.(C) Predicted GDF8 structure with the illustration of peptide binding ofthe human anti-myostatin antibodies. GDF8 structure was generated usingSWISS-MODEL; a fully automated protein structure homology-modelingserver, accessible via the ExPASy web server, or from the programDeepView (Swiss Pdb-Viewer). The first seven amino acids were missingfrom the predicted structure. Mature GDF8 is a homodimer protein. Onlyone subunit is shown. The antibodies disclosed herein bind to GDF8 as ahomodimer. As shown in the Figure, human monoclonal antibodies2_(—)43_(—)1, 2_(—)112_(—)1, and 2_(—)177_(—)1 bind to both peptide 1and 5. Peptides 1 and 5 are proximal spatially but not in primarystructure.

FIG. 7 shows the amino acid sequences of peptides 1-11 and correspondingsequence identifiers (SEQ ID NOs 103-113).

FIG. 8 depicts epitope binning. Epitope binding of the humananti-myostatin antibodies of the invention was mapped bycross-competition experiments using a BIACORE® 3000 instrument.Antibodies are depicted as labeled boxes. Antibodies in one circlecompete with antibodies in overlapping circles. For instance, antibody1_(—)46_(—)1 competes with antibody 1_(—)136_(—)3, but antibody1_(—)46_(—)1 does not compete with antibody 1_(—)268_(—)1. When two ormore antibodies are in the same circle their respective binding cannotbe distinguished.

FIG. 9 shows the results of an immunoprecipitation study to test theability of human anti-myostatin antibodies of the invention to pull downmature GDF8 and mature GDF8/propeptide latent complex. Conditionedmedium containing 293T cells transfected with GDF8 was used. As shown inthe Figure, antibodies 2_(—)112_(—)1, 2_(—)43_(—)1, and 2_(—)177_(—)1pulled down mature GDF8, mature GDF8/propeptide complex, and unprocessedGDF8; antibody 1_(—)66_(—)1 pulled down mature GDF8 and matureGDF8/propeptide complex; no other antibodies pulled down mature GDF8,propeptide, or unprocessed GDF8. Lane 4 is the 1/10 medium loadingcontrol, and lane 7 is immunoprecipitation control with medium only.

FIG. 10 shows the results of an immunoprecipitation study to test theability of anti-myostatin antibodies of the invention to pull downmature GDF8 from mouse serum. As shown is the Figure, antibodies2_(—)112_(—)1, 2_(—)43_(—)1, and 2_(—)177_(—)1 pulled down mature GDF8from the mouse serum, whereas antibodies 1_(—)116_(—)1, and 1_(—)66_(—)1could not.

FIG. 11 is a sequence alignment of mature human GDF8 and GDF11. MatureGDF8 (SEQ ID NO: 89) and GDF11 (SEQ ID NO: 90) share approximately 90%identical amino acid sequences. Both mature GDF8 and GDF11 formhomodimers. Both mature GDF8 or GDF11 have nine cysteines, which formfour internal disulfide bonds and one intermolecular disulfide bond. Asshown in the Figure, cysteines that form a disulfide bond with eachother are labeled with the same dots or plus. One cysteine (cys73) thatforms intermolecular disulfide bond was labeled with a star. GDF8structure was predicted using SWISS-MODEL (see FIG. 6C).

FIG. 12 is a table summarizing in vitro assay data.

FIG. 13 is a table summarizing gene usage, epitope binning and peptidebinding.

FIGS. 14 and 15 show the effects on muscle mass in mice following invivo treatment with anti-myostatin antibodies as compared with vehiclefor the gastrocnemius-pantaris-sloeus (GPS), tibilalis anterior (TA) andquadriceps (Quads) muscles.

FIG. 16 shows the effects on muscle weight (A) and strength (B) in SCIDmice following in vivo treatment with an anti-myostatin antibody(2_(—)112_K).

FIG. 17 illustrates the effects on muscle weight (A; B; C and D) in SCIDmice following in vivo treatment with varying doses of an anti-myostatinantibody (2_(—)112_K).

FIG. 18 is a dose (A) and concentration (B) response analysis of varyingdoses of an anti-myostatin antibody (2_(—)112_K) and their effects onmuscle weight.

FIG. 19 is an alignment of the heavy (A) and light chain (B) variableregions of antibodies 2_(—)112_(—)1 (heavy chain: SEQ ID NO:77; lightchain: SEQ ID NO:79) and 2_(—)112_K (heavy chain: SEQ ID NO:118; lightchain: SEQ ID NO:120).

FIG. 20 show the effects on muscle mass (A) and adipose mass (B) in micefollowing in vivo treatment with cortisone with an anti-myostatinantibody (2_(—)112_K) as compared with vehicle for thegastrocnemius-pantaris-sloeus (GPS), tibilalis anterior (TA) andquadriceps (Quads) muscles (in 20A) and the inguinal, epididymal andabdominla fat pad masses (in 20B).

DETAILED DESCRIPTION OF THE INVENTION Definitions and General Techniques

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclature used in connection with, and techniques of, cell and tissueculture, molecular biology, immunology, microbiology, genetics andprotein and nucleic acid chemistry and hybridization described hereinare those well known and commonly used in the art.

The methods and techniques of the present invention are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989) and Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates (1992), and Harlow and LaneAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1990), incorporated herein by reference.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The nomenclature used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well known and commonly used in the art. Standardtechniques are used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric.

The term “isolated protein”, “isolated polypeptide” or “isolatedantibody” is a protein, polypeptide or antibody that by virtue of itsorigin or source of derivation (1) is not associated with naturallyassociated components that accompany it in its native state, (2) is freeof other proteins from the same species, (3) is expressed by a cell froma different species, or (4) does not occur in nature. Thus, apolypeptide that is chemically synthesized or synthesized in a cellularsystem different from the cell from which it naturally originates willbe “isolated” from its naturally associated components. A protein mayalso be rendered substantially free of naturally associated componentsby isolation, using protein purification techniques well known in theart.

In various embodiments an isolated anti-myostatin antibody is one thathas been protein A-purified (see, e.g., Example XVI), one that has beensynthesized by a hybridoma or other cell line in vitro, and/or a humananti-myostatin antibody derived from a transgenic mouse.

A protein or polypeptide is “substantially pure,” “substantiallyhomogeneous,” or “substantially purified” when at least about 60 to 75%of a sample exhibits a single species of polypeptide. The polypeptide orprotein may be monomeric or multimeric. A substantially pure polypeptideor protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/Wof a protein sample, more usually about 95%, and preferably will be over99% pure. Protein purity or homogeneity may be indicated by a number ofmeans well known in the art, such as polyacrylamide gel electrophoresisof a protein sample, followed by visualizing a single polypeptide bandupon staining the gel with a stain well known in the art. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art for purification.

The term “non-human transgenic organism” as used herein refers to anynon-human transgenic individual living thing, including a non-humantransgenic animal, plant, bacterium, protist, or fungus.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence. In some embodiments,fragments are at least 5, 6, 8 or 10 amino acids long. In otherembodiments, the fragments are at least 14, at least 20, at least 50, orat least 70, 80, 90, 100, 150 or 200 amino acids long.

The term “polypeptide analog” as used herein refers to a polypeptidethat comprises a segment that has substantial identity to a portion ofan amino acid sequence and that has at least one of the followingproperties: (1) specific binding to myostatin under suitable bindingconditions, (2) ability to inhibit or activate myostatin. Typically,polypeptide analogs comprise a conservative amino acid substitution (orinsertion or deletion) with respect to the native sequence. Analogstypically are at least 20 or 25 amino acids long, preferably at least50, 60, 70, 80, 90, 100, 150 or 200 amino acids long or longer, and canoften be as long as a full-length polypeptide. Some embodiments of theinvention include polypeptide fragments or polypeptide analog antibodieswith 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17substitutions from the germline amino acid sequence.

In certain embodiments, amino acid substitutions to an anti-myostatinantibody or antigen-binding portion thereof are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, and (4) conferor modify other physicochemical or functional properties of suchanalogs, but still retain specific binding to myostatin. Analogs caninclude various muteins of a sequence other than the normally-occurringpeptide sequence. For example, single or multiple amino acidsubstitutions, preferably conservative amino acid substitutions, may bemade in the normally-occurring sequence, preferably in the portion ofthe polypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence; e.g., a replacementamino acid should not alter the anti-parallel β-sheet that makes up theimmunoglobulin binding domain that occurs in the parent sequence, ordisrupt other types of secondary structure that characterizes the parentsequence. In general, glycine and proline would not be used in ananti-parallel β-sheet. Examples of art-recognized polypeptide secondaryand tertiary structures are described in Proteins, Structures andMolecular Principles (Creighton, Ed., W.H. Freeman and Company, New York(1984)); Introduction to Protein Structure (C. Branden and J. Tooze,eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al.,Nature 354:105 (1991), incorporated herein by reference.

Non-peptide analogs are commonly used in the pharmaceutical industry asdrugs with properties analogous to those of the template peptide. Thesetypes of non-peptide compound are termed “peptide mimetics” or“peptidomimetics.” Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber andFreidinger, TINS p. 392 (1985); and Evans et al., J. Med. Chem. 30:1229(1987), incorporated herein by reference. Such compounds are oftendeveloped with the aid of computerized molecular modeling. Peptidemimetics that are structurally similar to therapeutically usefulpeptides may be used to produce an equivalent therapeutic orprophylactic effect. Generally, peptidomimetics are structurally similarto a paradigm polypeptide (i.e., a polypeptide that has a desiredbiochemical property or pharmacological activity), such as a humanantibody, but have one or more peptide linkages optionally replaced by alinkage selected from the group consisting of: —CH₂NH—, —CH₂S—,—CH₂—CH₂—, —CH═CH-(cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, bymethods well known in the art. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) may also be used to generate morestable peptides. In addition, constrained peptides comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo andGierasch, Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference); for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

Where an “antibody” is referred to herein with respect to the invention,it is normally understood that an antigen-binding portion thereof mayalso be used. An antigen-binding portion competes with the intactantibody for specific binding. See generally, Fundamental Immunology,Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated byreference in its entirety for all purposes). Antigen-binding portionsmay be produced by recombinant DNA techniques or by enzymatic orchemical cleavage of intact antibodies. In some embodiments,antigen-binding portions include Fab, Fab′, F(ab′)₂, Fd, Fv, dAb, andcomplementarity determining region (CDR) fragments, single-chainantibodies (scFv), chimeric antibodies, diabodies and polypeptides thatcontain at least a portion of an antibody that is sufficient to conferspecific antigen binding to the polypeptide.

From N-terminus to C-terminus, both the mature light and heavy chainvariable domains comprise, sequentially, the regions FR1, CDR1, FR2,CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domainherein is in accordance with the definitions of Kabat, Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md. (1987 and 1991)), Chothia & Lesk, J. Mol. Biol.196:901-917 (1987) or Chothia et al., Nature 342:878-883 (1989).

As used herein, an antibody that is referred to by number is the same asa monoclonal antibody that is obtained from the hybridoma of the samenumber. For example, monoclonal antibody 2_(—)112 is the same antibodyas one obtained from hybridoma 2_(—)112, or a subclone thereof, such as2_(—)112_(—)1, 2_(—)112_(—)2, and the like. The only exception is1_(—)136_(—)3 which is a different hybridoma from subclones1_(—)136_(—)1 and 1_(—)136_(—)2.

As used herein, a Fd fragment means an antibody fragment that consistsof the V_(H) and C_(H)1 domains; an Fv fragment consists of the V_(L)and V_(H) domains of a single arm of an antibody; and a dAb fragment(Ward et al., Nature 341:544-546 (1989)) consists of a V_(H) domain.

In some embodiments, the antibody is a single-chain antibody (scFv) inwhich V_(L) and V_(H) domains are paired to form a monovalent moleculesvia a synthetic linker that enables them to be made as a single proteinchain. (Bird et al., Science 242:423-426 (1988) and Huston et al., Proc.Natl. Acad. Sci. USA 85:5879-5883 (1988).) In some embodiments, theantibodies are diabodies, i.e., are bivalent antibodies in which V_(H)and V_(L) domains are expressed on a single polypeptide chain, but usinga linker that is too short to allow for pairing between the two domainson the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites. (See e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA90:6444-6448 (1993), and Poljak R. J. et al., Structure 2:1121-1123(1994).) In some embodiments, one or more CDRs from an antibody of theinvention may be incorporated into a molecule either covalently ornoncovalently to make it an immunoadhesin that specifically binds tomyostatin. In such embodiments, the CDR(s) may be incorporated as partof a larger polypeptide chain, may be covalently linked to anotherpolypeptide chain, or may be incorporated noncovalently. Further, theframework regions (FRs) may be derived from one of the anti-myostatinantibodies from which one or more of the CDRs are taken or from one ormore different human antibodies.

In embodiments having one or more binding sites, the binding sites maybe identical to one another or may be different.

As used herein, the term “human antibody” means any antibody in whichthe variable and constant domain sequences are human sequences. The termencompasses antibodies with sequences derived from (i.e., that utilize)human genes, but which have been changed, e.g. to decrease possibleimmunogenicity, increase affinity, eliminate cysteines that might causeundesirable folding, etc. The term encompasses such antibodies producedrecombinantly in non-human cells, which might impart glycosylation nottypical of human cells. These antibodies may be prepared in a variety ofways, as described below.

The term “chimeric antibody” as used herein means an antibody thatcomprises regions from two or more different antibodies. In oneembodiment, one or more of the CDRs of the chimeric antibody are derivedfrom a human anti-myostatin antibody. In another embodiment, all of theCDRs are derived from a human anti-myostatin antibodies. In anotherembodiment, the CDRs from more than one human anti-myostatin antibodiesare combined in a chimeric antibody. For instance, a chimeric antibodymay comprise a CDR1 from the light chain of a first human anti-myostatinantibody, a CDR2 from the light chain of a second human anti-myostatinantibody and a CDR3 from the light chain of a third human anti-myostatinantibody, and CDRs from the heavy chain may be derived from one or moreother anti-myostatin antibodies. Further, the framework regions may bederived from one of the anti-myostatin antibodies from which one or moreof the CDRs are taken or from one or more different human antibodies.

In some embodiments, a chimeric antibody of the invention is a humanizedanti-myostatin antibody. A humanized anti-myostatin antibody of theinvention comprises the amino acid sequence of one or more frameworkregions and/or the amino acid sequence from at least a portion of theconstant region of one or more human anti-myostatin antibodies of theinvention and further comprises sequences derived from a non-humananti-myostatin antibody, for example CDR sequences.

Fragments or analogs of antibodies or immunoglobulin molecules can bereadily prepared by those of ordinary skill in the art following theteachings of this specification. Preferred amino- and carboxy-termini offragments or analogs occur near boundaries of functional domains.Structural and functional domains can be identified by comparison of thenucleotide and/or amino acid sequence data to public or proprietarysequence databases. Preferably, computerized comparison methods are usedto identify sequence motifs or predicted protein conformation domainsthat occur in other proteins of known structure and/or function. Methodsto identify protein sequences that fold into a known three-dimensionalstructure are known. See Bowie et al., Science 253:164 (1991).

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIACORE® system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Johnsson U. et al., Ann. Biol. Clin. 51:19-26(1993); Jonsson U. et al., Biotechniques 11:620-627 (1991); Jonsson B.et al., J. Mol. Recognit. 8:125-131 (1995); and Johnsson B. et al.,Anal. Biochem. 198:268-277 (1991).

The term “K_(D)” means the equilibrium dissociation constant of aparticular antibody-antigen interaction.

The term “off rate” means the dissociation rate constant of a particularantibody-antigen interaction.

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor or otherwise interactingwith a molecule. Epitopic determinants generally consist of chemicallyactive surface groupings of molecules such as amino acids orcarbohydrate or sugar side chains and generally have specific threedimensional structural characteristics, as well as specific chargecharacteristics. An epitope may be “linear” or “conformational.” In alinear epitope, all of the points of interaction between the protein andthe interacting molecule (such as an antibody) occur linearly along theprimary amino acid sequence of the protein. In a conformational epitope,the points of interaction occur across amino acid residues on theprotein that are separated from one another. An antibody is said tospecifically bind an antigen when the dissociation constant is ≦1 mM,preferably ≦100 nM and most preferably ≦10 nM. In certain embodiments,the K_(D) is 1 pM to 500 pM. In other embodiments, the K_(D) is between500 pM to 1 μM. In other embodiments, the K_(D) is between 1 μM to 100nM. In other embodiments, the K_(D) is between 100 mM to 10 nM. Once adesired epitope on an antigen is determined, it is possible to generateantibodies to that epitope, e.g., using the techniques described in thepresent invention. Alternatively, during the discovery process, thegeneration and characterization of antibodies may elucidate informationabout desirable epitopes. From this information, it is then possible tocompetitively screen antibodies for binding to the same epitope. Anapproach to achieve this is to conduct cross-competition studies to findantibodies that competitively bind with one another, e.g., theantibodies compete for binding to the antigen. A high throughput processfor “binning” antibodies based upon their cross-competition is describedin International Patent Application No. WO 03/48731.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), incorporated herein by reference.

The term “polynucleotide” as referred to herein means a polymeric formof nucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms.

The term “isolated polynucleotide” as used herein means a polynucleotideof genomic, cDNA, or synthetic origin or some combination thereof, whichby virtue of its origin the “isolated polynucleotide” (1) is notassociated with all or a portion of a polynucleotides with which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide to which it is not linked in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “naturally occurring nucleotides” as used herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” as used herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See e.g., LaPlanche et al., Nucl. AcidsRes. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984);Stein et al., Nucl. Acids Res. 16:3209 (1988); Zon et al., Anti-CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues; APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); U.S. Pat. No. 5,151,510; Uhlmann andPeyman, Chemical Reviews 90:543 (1990), the disclosures of which arehereby incorporated by reference. An oligonucleotide can include a labelfor detection, if desired.

“Operably linked” sequences include both expression control sequencesthat are contiguous with the gene of interest and expression controlsequences that act in trans or at a distance to control the gene ofinterest. The term “expression control sequence” as used herein meanspolynucleotide sequences that are necessary to effect the expression andprocessing of coding sequences to which they are ligated. Expressioncontrol sequences include appropriate transcription initiation,termination, promoter and enhancer sequences; efficient RNA processingsignals such as splicing and polyadenylation signals; sequences thatstabilize cytoplasmic mRNA; sequences that enhance translationefficiency (i.e., Kozak consensus sequence); sequences that enhanceprotein stability; and when desired, sequences that enhance proteinsecretion. The nature of such control sequences differs depending uponthe host organism; in prokaryotes, such control sequences generallyinclude promoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequences” is intended to include, at a minimum, all components whosepresence is essential for expression and processing, and can alsoinclude additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences.

The term “vector”, as used herein, means a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked. Insome embodiments, the vector is a plasmid, i.e., a circular doublestranded piece of DNA into which additional DNA segments may be ligated.In some embodiments, the vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. In some embodiments,the vectors are capable of autonomous replication in a host cell intowhich they are introduced (e.g., bacterial vectors having a bacterialorigin of replication and episomal mammalian vectors). In otherembodiments, the vectors (e.g., non-episomal mammalian vectors) can beintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “expressionvectors”).

The term “recombinant host cell” (or simply “host cell”), as usedherein, means a cell into which a recombinant expression vector has beenintroduced. It should be understood that “recombinant host cell” and“host cell” mean not only the particular subject cell but also theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof in accordance with the invention selectively hybridize tonucleic acid strands under hybridization and wash conditions thatminimize appreciable amounts of detectable binding to nonspecificnucleic acids. “High stringency” or “highly stringent” conditions can beused to achieve selective hybridization conditions as known in the artand discussed herein. One example of “high stringency” or “highlystringent” conditions is the incubation of a polynucleotide with anotherpolynucleotide, wherein one polynucleotide may be affixed to a solidsurface such as a membrane, in a hybridization buffer of 6×SSPE or SSC,50% formamide, 5×Denhardt's reagent, 0.5% SDS, 100 μg/ml denatured,fragmented salmon sperm DNA at a hybridization temperature of 42° C. for12-16 hours, followed by twice washing at 55° C. using a wash buffer of1×SSC, 0.5% SDS. See also Sambrook et al., supra, pp. 9.50-9.55.

The term “percent sequence identity” in the context of nucleic acidsequences means the residues in two sequences that are the same whenaligned for maximum correspondence. The length of sequence identitycomparison may be over a stretch of at least about nine nucleotides,usually at least about 18 nucleotides, more usually at least about 24nucleotides, typically at least about 28 nucleotides, more typically atleast about 32 nucleotides, and preferably at least about 36, 48 or morenucleotides. There are a number of different algorithms known in the artwhich can be used to measure nucleotide sequence identity. For instance,polynucleotide sequences can be compared using FASTA, Gap or BESTFIT®,which are programs in Wisconsin Package Version 10.0, Genetics ComputerGroup (GCG), Madison, Wis. FASTA, which includes, e.g., the programsFASTA2 and FASTA3, provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol.132:185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996);Pearson, J. Mol. Biol. 276:71-84 (1998); incorporated herein byreference). Unless otherwise specified, default parameters for aparticular program or algorithm are used. For instance, percent sequenceidentity between nucleic acid sequences can be determined using FASTAwith its default parameters (a word size of 6 and the NOPAM factor forthe scoring matrix) or using Gap with its default parameters as providedin GCG Version 6.1, incorporated herein by reference.

A reference to a nucleotide sequence encompasses its complement unlessotherwise specified. Thus, a reference to a nucleic acid having aparticular sequence should be understood to encompass its complementarystrand, with its complementary sequence.

As used herein, the terms “percent sequence identity” and “percentsequence homology” are used interchangeably.

The term “substantial similarity” or “substantial sequence similarity,”when referring to a nucleic acid or fragment thereof, means that whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 85%, preferably at leastabout 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99%of the nucleotide bases, as measured by any well-known algorithm ofsequence identity, such as FASTA, BLAST or Gap, as discussed above.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT® using default gap weights as supplied with the programs,share at least 70%, 75% or 80% sequence identity, preferably at least90% or 95% sequence identity, and more preferably at least 97%, 98% or99% sequence identity. In certain embodiments, residue positions thatare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chainR group with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well-known to thoseof skill in the art. See, e.g., Pearson, Methods Mol. Biol. 243:307-31(1994). Examples of groups of amino acids that have side chains withsimilar chemical properties include 1) aliphatic side chains: glycine,alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl sidechains: serine and threonine; 3) amide-containing side chains:asparagine and glutamine; 4) aromatic side chains: phenylalanine,tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, andhistidine; 6) acidic side chains: aspartic acid and glutamic acid; and7) sulfur-containing side chains: cysteine and methionine. Conservativeamino acids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine.

Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al., Science 256:1443-45 (1992), incorporated herein by reference. A“moderately conservative” replacement is any change having a nonnegativevalue in the PAM250 log-likelihood matrix.

Sequence identity for polypeptides is typically measured using sequenceanalysis software. Protein analysis software matches sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG contains programs such as “Gap” and “BESTFIT®” whichcan be used with default parameters as specified by the programs todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and a mutein thereof. See,e.g., GCG Version 6.1 (University of Wisconsin, WI). Polypeptidesequences also can be compared using FASTA using default or recommendedparameters, see GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3)provides alignments and percent sequence identity of the regions of thebest overlap between the query and search sequences (Pearson, MethodsEnzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219(2000)). Another preferred algorithm when comparing a sequence of theinvention to a database containing a large number of sequences fromdifferent organisms is the computer program BLAST, especially blastp ortblastn, using default parameters as supplied with the programs. See,e.g., Altschul et al., J. Mol. Biol. 215:403-410 (1990); Altschul etal., Nucleic Acids Res. 25:3389-402 (1997).

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, usually at least about 20residues, more usually at least about 24 residues, typically at leastabout 28 residues, and preferably more than about 35 residues. Whensearching a database containing sequences from a large number ofdifferent organisms, it is preferable to compare amino acid sequences.

As used herein, the terms “label” or “labeled” refers to incorporationof another molecule in the antibody. In one embodiment, the label is adetectable marker, e.g., incorporation of a radiolabeled amino acid orattachment to a polypeptide of biotinyl moieties that can be detected bymarked avidin (e.g., streptavidin containing a fluorescent marker orenzymatic activity that can be detected by optical or colorimetricmethods). In another embodiment, the label or marker can be therapeutic,e.g., a drug conjugate or toxin. Various methods of labelingpolypeptides and glycoproteins are known in the art and may be used.Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescentmarkers, biotinyl groups, predetermined polypeptide epitopes recognizedby a secondary reporter (e.g., leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags),magnetic agents, such as gadolinium chelates, toxins such as pertussistoxin, TAXOL® (paclitaxel), cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. In some embodiments, labelsare attached by spacer arms of various lengths to reduce potentialsteric hindrance.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

Human Anti-Myostatin Antibodies and Characterization Thereof

The invention herein provides anti-myostatin antibodies. In someembodiments, the antibodies are human. In other embodiments, theantibodies are humanized. In some embodiments, human anti-myostatinantibodies are produced by immunizing a non-human transgenic animal,e.g., a rodent, whose genome comprises human immunoglobulin genes sothat the transgenic animal produces human antibodies.

An anti-myostatin antibody of the invention can comprise a human kappaor a human lambda light chain or an amino acid sequence derivedtherefrom. In some embodiments comprising a kappa light chain, the lightchain variable domain (V_(L)) utilizes a human A30, A3 or L2 V_(K) gene

In some embodiments, the V_(L) of the anti-myostatin antibody comprisesone or more amino acid substitutions, deletions or insertions(additions) relative to the germline V_(K) amino acid sequence. In someembodiments, the V_(L) of the anti-myostatin antibody comprises 1, 2, 3,4 or 5 amino acid substitutions relative to the germline V_(K) aminoacid sequence. In some embodiments, one or more of the substitutionsfrom germline is in the CDR regions of the light chain. In someembodiments, the V_(K) amino acid substitutions relative to germline areat one or more of the same positions as the substitutions relative togermline found in any one or more of the V_(L) of the antibodiesprovided herein. For example, the V_(L) of an anti-myostatin antibody ofthe invention may contain one or more of the amino acid substitutionscompared to germline found in the V_(L) of antibody 1_(—)257_(—)1. Therealso may be one or more amino acid substitutions compared to germlinefound in the V_(L) of antibody 1_(—)116_(—)1, which utilizes the sameV_(K) gene as antibody 1_(—)257_(—)1. In some embodiments, the aminoacid changes are at one or more of the same positions, but involve adifferent substitution than in the reference antibody. In all cases, therecitation of an antibody clone with dashes is equivalent to the sameantibody clone with dots (e.g., 1_(—)257_(—)1=1.257.1).

In some embodiments, amino acid substitutions relative to germline occurat one or more of the same positions as substitutions from germline inany of the V_(L) of antibodies 1_(—)116_(—)1, 1_(—)136_(—)3,1_(—)257_(—)1, 1_(—)46_(—)1, 2_(—)112_(—), 1_(—)314_(—)1, 1_(—)66_(—)1,2_(—)43_(—)1, 2_(—)177_(—)1, 1_(—)132_(—)1, 1_(—)268_(—)1,1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I; 1_(—)314_(—)1H-T92A;1_(—)46_(—)1H-L81M; 2_(—)112_(—)1H-I12V; 2_(—)112_(—)1L-F58I;2_(—)112_(—)1L-I85V; 2_(—)112_(—)1H-L81M, L-F58I; 2_(—)112_(—)1H-L81M,L-I85V; or 2_(—)112_(—)1H-L81M, L-F58I, I85V, but the substitutions mayrepresent conservative amino acid substitutions at such position(s)relative to the amino acid in the reference antibody. For example, if aparticular position in one of these antibodies is changed relative togermline and is glutamate, one may substitute aspartate at thatposition. Similarly, if an amino acid substitution compared to germlinein an exemplified antibody is serine, one may conservatively substitutethreonine for serine at that position. Conservative amino acidsubstitutions are discussed supra, throughout this application.

In some embodiments, the anti-myostatin antibody comprises a light chainamino acid sequence selected from the group consisting of SEQ ID NOS: 4,8, 12, 16, 20, 24, 28, 32, 36, 40 and 44. In other embodiments, thelight chain comprises the light chain amino acid sequence of antibody1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I; 2 _(—)112_(—)1L-F58I; 2_(—)112_(—)1L-I85V or 2_(—)112_(—)1L-F58I, I85V. In some embodiments,the light chain of the human anti-myostatin antibody comprises the V_(L)amino acid sequence of antibody 1_(—)116_(—)1 (SEQ ID NO: 4);1_(—)136_(—)3 (SEQ ID NO: 12); 1_(—)257_(—)1 (SEQ ID NO: 16);1_(—)46_(—)1 (SEQ ID NO: 24); 2_(—)112_(—)1 (SEQ ID NO: 28);1_(—)314_(—)1 (SEQ ID NO: 20); 1_(—)66_(—)1 (SEQ ID NO: 36);2_(—)43_(—)1 (SEQ ID NO: 44); 2_(—)177_(—)1 (SEQ ID NO: 40);1_(—)132_(—)1 (SEQ ID NO: 8); or 1_(—)268_(—)1 (SEQ ID NO: 32) or saidamino acid sequence having up to 1, 2, 3, 4 or 5 conservative amino acidsubstitutions and/or a total of up to 3 non-conservative amino acidsubstitutions. In other embodiments the light chain of the humananti-myostatin antibody comprises the V_(L) amino acid sequence ofantibody 1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I; 2_(—)112_(—)1L-F58I;2 _(—)112_(—)1L-I85V or 2_(—)112_(—)1L-F58I, I85V. In some embodiments,the light chain comprises the amino acid sequence from the beginning ofthe CDR1 to the end of the CDR3 of any one of the foregoing antibodies.

In some embodiments, the light chain may comprise the amino acidsequences of CDR1, CDR2 and CDR3 regions independently selected from thelight chain CDR1, CDR2 and CDR3 regions, respectively, of two or moremonoclonal antibodies selected from 1_(—)116_(—)1 (SEQ ID NO: 4);1_(—)136_(—)3 (SEQ ID NO: 12); 1_(—)257_(—)1 (SEQ ID NO: 16);1_(—)46_(—)1 (SEQ ID NO: 24); 2_(—)112_(—)1 (SEQ ID NO: 28);1_(—)314_(—)1 (SEQ ID NO: 20); 1_(—)66_(—)1 (SEQ ID NO: 36);2_(—)43_(—)1 (SEQ ID NO: 44); 2_(—)177_(—)1 (SEQ ID NO: 40);1_(—)132_(—)1 (SEQ ID NO: 8); or 1_(—)268_(—)1 (SEQ ID NO: 32), or saidCDR regions each having less than 3 or less than 2 conservative aminoacid substitutions and/or a total of three or fewer non-conservativeamino acid substitutions.

In certain embodiments, the light chain of the anti-myostatin antibodycomprises the amino acid sequences of the light chain CDR1, CDR2 andCDR3 regions of an antibody selected from 1_(—)116_(—)1 (SEQ ID NO: 4);1_(—)136_(—)3 (SEQ ID NO: 12); 1_(—)257_(—)1 (SEQ ID NO: 16);1_(—)46_(—)1 (SEQ ID NO: 24); 2_(—)112_(—)1 (SEQ ID NO: 28);1_(—)314_(—)1 (SEQ ID NO: 20); 1_(—)66_(—)1 (SEQ ID NO: 36);2_(—)43_(—)1 (SEQ ID NO: 44); 2_(—)177_(—)1 (SEQ ID NO: 40);1_(—)132_(—)1 (SEQ ID NO: 8); or 1_(—)268_(—)1 (SEQ ID NO: 32) or saidCDR regions each having less than 3 or less than 2 conservative aminoacid substitutions and/or a total of three or fewer non-conservativeamino acid substitutions.

With regard to the heavy chain, in some embodiments, the variable domain(V_(H)) utilizes a human V_(H) 1-02, V_(H) 3-21 or V_(H) 3-23 gene. Insome embodiments, the V_(H) sequence of the anti-myostatin antibodycontains one or more amino acid substitutions, deletions or insertions(additions), collectively “mutations”, relative to the germline V_(H)amino acid sequence. In some embodiments, the variable domain of theheavy chain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 mutations fromthe germline V_(H) amino acid sequence. In some embodiments, themutation(s) are non-conservative substitutions compared to the germlineamino acid sequence. In some embodiments, the mutations are in the CDRregions of the heavy chain.

In some embodiments, amino acid substitutions are at one or more of thesame positions as the substitutions from germline in any one or more ofthe V_(H) of antibodies 1_(—)116_(—)1, 1_(—)136_(—)3, 1_(—)257_(—)1,1_(—)46_(—)1, 2_(—)112_(—), 1_(—)314_(—)1, 1_(—)66_(—)1, 2_(—)43_(—)1,2_(—)177_(—)1, 1_(—)132_(—)1, 1_(—)268_(—)1, 1_(—)116_(—)1L-Q45K;1_(—)257_(—)1L-L21I; 1_(—)314_(—)1H-T92A; 1_(—)46_(—)1H-L81M;2_(—)112_(—)1H-I12V; 2_(—)112_(—)1L-F58I; 2_(—)112_(—)1L-I85V;2_(—)112_(—)1H-L81M, L-F58I; 2_(—)112_(—)1H-L81M, L-I85V; or2_(—)112_(—)1H-L81M, L-F58I, I85V. In other embodiments, the amino acidchanges are at one or more of the same positions but involve a differentsubstitution than in the reference antibody.

In some embodiments, the heavy chain comprises an amino acid sequenceselected from the group consisting of SEQ ID NOS: 2, 6, 10, 14, 18, 22,26, 30, 34, 38 and 42. In other embodiments, the heavy chain comprisesthe heavy chain amino acid sequence of antibody 1_(—)314_(—)1H-T92A;1_(—)46_(—)1H-L81M; or 2_(—)112_(—)1H-I12V. In some embodiments, theheavy chain comprises the V_(H) amino acid sequence of antibody1_(—)116_(—)1 (SEQ ID NO: 2), 1_(—)136_(—)3 (SEQ ID NO: 10),1_(—)257_(—)1 (SEQ ID NO: 14), 1_(—)46_(—)1 (SEQ ID NO: 22),2_(—)112_(—)1 (SEQ ID NO: 26), 1_(—)314_(—)1 (SEQ ID NO: 18),1_(—)66_(—)1 (SEQ ID NO: 34), 2_(—)43_(—)1 (SEQ ID NO: 42),2_(—)177_(—)1 (SEQ ID NO: 38), 1_(—)132_(—)1 (SEQ ID NO: 6) and1_(—)268_(—)1 (SEQ ID NO: 30); or said V_(H) amino acid sequence havingup to 1, 2, 3, 4, 6, 8, 9, 10 or 11 conservative amino acidsubstitutions and/or a total of up to 3 non-conservative amino acidsubstitutions. In other embodiments, the heavy chain comprises the V_(H)amino acid sequence of antibody 1_(—)314_(—)1H-T92A; 1_(—)46_(—)1H-L81M;or 2_(—)112_(—)1H-I12V. In some embodiments, the heavy chain comprisesthe amino acid sequence from the beginning of the CDR1 to the end of theCDR3 of any one of the foregoing antibodies.

In some embodiments, the heavy chain comprises the heavy chain CDR1,CDR2 and CDR3 regions of antibody 1_(—)116_(—)1, 1_(—)136_(—)3,1_(—)257_(—)1, 1_(—)46_(—)1, 2_(—)112_(—), 1_(—)314_(—)1, 1_(—)66_(—)1,2_(—)43_(—)1, 2_(—)177_(—)1, 1_(—)132_(—)1, 1_(—)268_(—)1,1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I; 1_(—)314_(—)1H-T92A;1_(—)46_(—)1H-L81M; 2_(—)112_(—)1H-I12V; 2_(—)112_(—)1L-F58I;2_(—)112_(—)1 L-I85V; 2_(—)112_(—)1H-L81M, L-F58I; 2 _(—)112_(—)1H-L81M,L-I85V; or 2_(—)112_(—)1H-L81M, L-F58I, I85V or said CDR regions eachhaving less than 8, less than 6, less than 4, or less than 3conservative amino acid substitutions and/or a total of three or fewernon-conservative amino acid substitutions.

In some embodiments, the heavy chain CDR regions are independentlyselected from the CDR regions of two or more antibodies selected fromantibodies 1_(—)116_(—)1, 1_(—)136_(—)3, 1_(—)257_(—)1, 1_(—)46_(—)1,2_(—)112_(—), 1_(—)314_(—)1, 1_(—)66_(—)1, 2_(—)43_(—)1, 2_(—)177_(—)1,1_(—)132_(—)1, 1_(—)268_(—)1, 1_(—)116_(—)1 L-Q45K; 1_(—)257_(—)1L-L21I; 1_(—)314_(—)1H-T92A; 1_(—)46_(—)1H-L81M; 2_(—)112_(—)1H-I12V;2_(—)112_(—)1L-F58I; 2_(—)112_(—)1L-I85V; 2_(—)112_(—)1H-L81M, L-F58I;2_(—)112_(—)1H-L81M, L-I85V; or 2_(—)112_(—)1H-L81M, L-F58I, I85V. Inanother embodiment, the antibody comprises a light chain as disclosedabove and a heavy chain as disclosed above. In a further embodiment, thelight chain CDRs and the heavy chain CDRs are from the same antibody.

In various embodiments, the anti-myostatin antibodies have thefull-length heavy chain and full length light chain amino acidsequence(s), the V_(H) and V_(L) amino acid sequences, the heavy chainCDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 amino acidsequences or the heavy chain amino acid sequence from the beginning ofthe CDR1 to the end of the CDR3 and the light chain amino acid sequencefrom the beginning of the CDR1 to the end of the CDR3 of ananti-myostatin antibody provided herein.

One type of amino acid substitution that may be made is to change one ormore cysteines in the antibody, which may be chemically reactive, toanother residue, such as, without limitation, alanine or serine. In oneembodiment, there is a substitution of a non-canonical cysteine. Thesubstitution can be made in a CDR or framework region of a variabledomain or in the constant domain of an antibody. In some embodiments,the cysteine is canonical.

Another type of amino acid substitution that may be made is to removepotential proteolytic sites in the antibody. Such sites may occur in aCDR or framework region of a variable domain or in the constant domainof an antibody. Substitution of cysteine residues and removal ofproteolytic sites may decrease the risk of heterogeneity in the antibodyproduct and thus increase its homogeneity. Another type of amino acidsubstitution is to eliminate asparagine-glycine pairs, which formpotential deamidation sites, by altering one or both of the residues.

In some embodiments, the C-terminal lysine of the heavy chain of theanti myostatin antibody of the invention is cleaved. In variousembodiments of the invention, the heavy and light chains of theanti-myostatin antibodies may optionally include a signal sequence.

In one aspect, the invention provides to eleven inhibitory humananti-myostatin monoclonal antibodies and the hybridoma cell lines thatproduce them. Table 1 lists the sequence identifiers (SEQ ID NOs) of thenucleic acids encoding the full-length heavy and light chains andvariable domain containing portions of those chains, and thecorresponding full-length deduced amino acid sequences.

TABLE 1 SEQUENCE IDENTIFIERS (SEQ ID NO) FULL LENGTH V DOMAIN CONTAININGPORTION Heavy Light Heavy Light Mab DNA PROTEIN DNA PROTEIN PROTEIN DNAPROTEIN DNA 1_116_1 1 2 3 4 45 46 47 48 1_132_1 5 6 7 8 49 50 51 521_136_3 9 10 11 12 53 54 55 56 1_257_1 13 14 15 16 57 58 59 60 1_314_117 18 19 20 65 66 67 68 1_46_1 21 22 23 24 69 70 71 72 2_112_1 25 26 2728 77 78 79 80 1_268_1 29 30 31 32 61 62 63 64 1_66_1 33 34 35 36 73 7475 76 2_177_1 37 38 39 40 81 82 83 84 2_43_1 41 42 43 44 85 86 87 882_112_K 114 115 116 117 118 119 120 121

The invention further provides heavy and/or light chain variants ofcertain of the above-listed human anti-myostatin antibodies, comprisingone or more amino acid modifications. To designate the variants, thefirst letter is the one letter symbol for the amino acid of thenaturally-occurring antibody chain, the number refers to the position ofthe amino acid (wherein position one is the N-terminal amino acid of theFRI), and the second letter is the one letter symbol for the variantamino acid.

The invention provides a light chain variant of monoclonal antibody1_(—)116_(—)1 called 1_(—)116_(—)1L-Q45K, which has a lysine at position45 of SEQ ID NO: 4.

Another light chain variant is 1_(—)257_(—)1 L-L21I, which has anisoleucine residue at position 21 of SEQ ID NO: 16.

A heavy chain varianty of monoclonal antibody of 1_(—)314_(—)1 called1_(—)314_(—)1H-T92A which has an alanine residue at position 92 of SEQID NO: 18.

The invention further provides a heavy chain variant of monoclonalantibody 1_(—)46_(—)1 called 1_(—)46_(—)1H-L81M, which has a methionineat position 81 of SEQ ID NO: 22.

The invention provides a heavy chain variant (I12V) and two light chainvariants (F581 and I85V) of monoclonal antibody 2_(—)112_(—)1. The heavychain variant called 2_(—)112_(—)1H-I12V has a valine at position 12 ofSEQ ID NO: 26. One light chain variant, 2_(—)112_(—)1L-F85I, has anisoleucine at position 85 of SEQ ID NO: 28. The other light chainvariant, called 2_(—)112_(—)1L-I85V, has a valine at position 85 of SEQID NO: 28. The invention also provides a light chain variant comprisingboth mutations, i.e., 2_(—)112_(—)1L-F85I, I85V. The invention alsoincludes antibodies comprising the heavy chain variant with either oneor both of the light chain mutations, i.e., 2_(—)112_(—)1H-I12V, L-F58I;2_(—)112_(—)1H-I12V, L-I85V; and 2_(—)112_(—)1H-I12V, L-F58I, I85V.

In still further embodiments, the invention includes antibodiescomprising variable domain amino acid sequences with more than 80%, morethan 85%, more than 90%, more than 95%, more than 96%, more than 97%,more than 98% or more than 99% sequence identity to a variable domainamino acid sequence of any of the above-listed human anti-myostatinantibodies

Class and Subclass of Anti-myostatin Antibodies

The class and subclass of anti-myostatin antibodies may be determined byany method known in the art. In general, the class and subclass of anantibody may be determined using antibodies that are specific for aparticular class and subclass of antibody. Such antibodies arecommercially available. The class and subclass can be determined byELISA, or Western Blot as well as other techniques. Alternatively, theclass and subclass may be determined by sequencing all or a portion ofthe constant domains of the heavy and/or light chains of the antibodies,comparing their amino acid sequences to the known amino acid sequencesof various class and subclasses of immunoglobulins, and determining theclass and subclass of the antibodies.

In some embodiments, the anti-myostatin antibody is a monoclonalantibody. The anti-myostatin antibody can be an IgG, an IgM, an IgE, anIgA, or an IgD molecule. In a preferred embodiment, the anti-myostatinantibody is an IgG and is an IgG1, IgG2, IgG3, IgG4 subclass. In anotherpreferred embodiment, the antibody is subclass IgG2.

Identification of Myostatin Epitopes Recognized by Anti-MyostatinAntibodies

The invention provides a human anti-myostatin monoclonal antibody thatbinds to myostatin and competes or cross-competes with and/or binds thesame epitope as: (a) an antibody selected from 1_(—)116_(—)1,1_(—)136_(—)3, 1_(—)257_(—)1, 1_(—)46_(—)1, 2_(—)112_(—), 1_(—)314_(—)1,1_(—)66_(—)1, 2_(—)43_(—)1, 2_(—)177_(—)1, 1_(—)132_(—)1, 1_(—)268_(—)1,1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I; 1_(—)314_(—)1H-T92A;1_(—)46_(—)1H-L81M; 2_(—)112_(—)1H-I12V; 2_(—)112_(—)1L-F58I;2_(—)112_(—)1 L-I85V; 2_(—)112_(—)1H-L81M, L-F58I; 2 _(—)112_(—)1H-L81M,L-I85V; or 2_(—)112_(—)1H-L81M, L-F58I, I85V; (b) an antibody thatcomprises a heavy chain variable domain having the amino acid sequenceof the V_(H) domain in any one of SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26,30, 34, 38, 42, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81 or 85, (c) anantibody that comprises a light chain variable domain having the aminoacid sequence of the V_(L) domain in any one of SEQ ID NOS: 4, 8, 12,16, 20, 24, 28, 32, 36, 40, 44, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83or 87 (d) an antibody that comprises both a heavy chain variable domainas defined in (b) and a light chain variable domain as defined in (c).

One can determine whether an antibody binds to the same epitope or crosscompetes for binding with an anti-myostatin antibody by using methodsknown in the art. In one embodiment, one allows the anti-myostatinantibody of the invention to bind to myostatin under saturatingconditions and then measures the ability of the test antibody to bind tomyostatin. If the test antibody is able to bind to myostatin at the sametime as the reference anti-myostatin antibody, then the test antibodybinds to a different epitope than the reference anti-myostatin antibody.However, if the test antibody is not able to bind to myostatin at thesame time, then the test antibody binds to the same epitope, anoverlapping epitope, or an epitope that is in close proximity to theepitope bound by the anti-myostatin antibody of the invention. Thisexperiment can be performed using ELISA, RIA, BIACORE®, or flowcytometry. To test whether an anti-myostatin antibody cross-competeswith another anti-myostatin antibody, one may use the competition methoddescribed above in two directions, i.e. determining if the knownantibody blocks the test antibody and vice versa. In a preferredembodiment, the experiment is performed using ELISA.

Inhibition of Myostatin Activity by Anti-Myostatin Antibody

One can identify anti-myostatin monoclonal antibodies that inhibitmyostatin binding using a number of assays. For example, neutralizinganti-myostatin antibodies can be identified by their ability to blockmyostatin-induced luciferase activity in A204 cells transfected with aSmad response elements/luciferase construct as described in Example III.Preferred anti-myostatin antibodies have an IC₅₀ of no more than 500 nM,250 nM, 100 nM, 75 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 1 nM, 0.5 nMor 0.1 nM.

One also can determine the ability of an anti-myostatin antibody toblock myostatin-induced Smad protein activation by contacting L6 ratmyoblast cells transfected with a Smad 2/3-binding element/betalactamase construct with an anti-myostatin antibody as described inExample IV. In various embodiments, the anti-myostatin antibody has anIC₅₀ in this assay of no more than 500 nM, 250 nM, 100 nM, 75 nM, 50 nM,40 nM, 30 nM, 20 nM, 10 nM, 1 nM, 0.5 nM or 0.1 nM.

Alternatively, neutralizing anti-myostatin anti-bodies can be identifiedby their ability to inhibit Smad 2 or 3 phosphorylation in a WesternBlot as described in Example V.

In other embodiments, an anti-myostatin antibody of the inventionmodulates the expression of genes associated with muscle cellproliferation and differentiation. Such modulation includes but is notlimited to decreasing expression of the cell cycle inhibitor P21 proteinand pro-apoptotic Bax protein, increasing phosphorylation of Rb, andincreasing expression of Cdk2. In some embodiments, antagonistanti-myostatin antibodies of the invention increase the expression ofMyoD, myogenin and Myf5. The effect of an anti-myostatin antibody ongene expression can be determined using any of a number of routinetechniques. (See e.g., Example VI).

In some embodiments, a neutralizing anti-myostatin antibody of theinvention enhances myoblast differentiation. The ability of an antibodyto enhance such proliferation and differentiation can be determined byassays using, e.g., C2C12 cells as described in Example VII.

Competitive Vs Non-Competitive Human Anti-Myostatin Antibodies

Human anti-myostatin antibodies of the invention can be categorized ascompetitive and non-competitive with other inhibitory myostatin bindingproteins using immunoprecipitation experiments. For example, thepropeptide, which forms a complex with mature GDF8 is an inhibitoryprotein. Conditioned medium from 293T cells expressing GDF8 containsmature GDF8, mature GDF8/propeptide complex and unprocessed GDF8.Immunoprecipitation studies were conducted to test the binding of thehuman anti-myostatin antibodies in the invention to matureGDF8/propetide complex using this conditioned medium. As described inExample X, antibodies 2_(—)112_(—)1, 2_(—)43_(—)1 and 2_(—)177_(—)1bound and immunoprecipitated mature GDF8, mature GDF8/propeptidecomplex, and unprocessed GDF8. Antibody 1_(—)66_(—)1 bound andimmunoprecipitated mature GDF8 and mature GDF8/propeptide complex. Noneof the other antibodies immunoprecipitated mature GDF8, propeptide, orunprocessed GDF8. Antibodies 2_(—)112_(—)1, 2_(—)43_(—)1 and2_(—)177_(—)1, thus, are non-competitive antibodies, and antibody1_(—)66_(—)1 also is a non-competitive antibody but one that binds to adifferent epiptope of myostatin. A non-competitive neutralizingantibody, i.e., one that binds myostatin in the presence of otherinhibitory proteins, will have better in vivo efficacy than acompetitive antibody.

In other embodiments, an anti-myostatin antibody of the inventionimmunoprecipitates mature GDF8 from mouse serum. As described in ExampleXI, monoclonal antibodies 2_(—)112, 2_(—)43_(—)1 and 2_(—)177 pull downmore mature GDF8 than 1_(—)116_(—)1 and 1_(—)66_(—)1.

Species Specificity and Molecular Selectivity

In another aspect of the invention, the anti-myostatin antibodiesdemonstrate both species specificity and molecular selectivity. In someembodiments, the anti-myostatin antibody binds to human, mouse, Rattusnorvegicus (Norway rat), cynomolgus macaque (monkey), Macacafascicularis (crab-eating macaque), Meleagris gallopavo (turkey), Susscrofa (pig), Gallus gallus (chicken), Gallus gallus (chicken), Canisfamiliaris (dog), Equus caballus (horse), Coturnix chinensis (Quail),Coturnix coturnix (common quail), and Columba livia (domestic pigeon)myostatin. Following the teachings of the specification, one maydetermine the species specificity for the anti-myostatin antibody usingmethods well known in the art. For instance, one may determine thespecies specificity using Western blot, surface plasmon resonance, e.g.,BIACORE®, ELISA, immunoprecipitation or RIA.

In other embodiments, the anti-myostatin antibody has a selectivity formyostatin over GDF11 of at least 50-fold or at least 100-fold. GDF11 isa close family member of myostatin that shares ninety percent identityin its mature protein. In some embodiments, the anti-myostatin antibodydoes not exhibit any appreciable specific binding to any other proteinother than myostatin. One can determine the selectivity of theanti-myostatin antibody for myostatin using methods well known in theart following the teachings of the specification. For instance one candetermine the selectivity using Western blot, flow cytometry, ELISA,immunoprecipitation or RIA.

In other embodiments of the invention, the anti-myostatin antibody doesnot have this high degree of selectivity for GDF8 over GDF11.

Methods of Producing Antibodies and Antibody Producing Cell LinesImmunization

In some embodiments, human antibodies are produced by immunizing anon-human, transgenic animal comprising within its genome some or all ofhuman immunoglobulin heavy chain and light chain loci with a myostatinantigen. In a preferred embodiment, the non-human animal is a XENOMOUSE®animal. (Abgenix, Inc., Fremont, Calif.).

XENOMOUSE® mice are engineered mouse strains that comprise largefragments of human immunoglobulin heavy chain and light chain loci andare deficient in mouse antibody production. See, e.g., Green et al.,Nature Genetics 7:13-21 (1994) and U.S. Pat. Nos. 5,916,771, 5,939,598,5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, 6,130,364,6,162,963 and 6,150,584. See also WO 91/10741, WO 94/02602, WO 96/34096,WO 96/33735, WO 98/16654, WO 98/24893, WO 98/50433, WO 99/45031, WO99/53049, WO 00/09560, and WO 00/037504.

In another aspect, the invention provides a method for makinganti-myostatin antibodies from non-human, non-mouse animals byimmunizing non-human transgenic animals that comprise humanimmunoglobulin loci with a myostatin antigen. One can produce suchanimals using the methods described in the above-cited documents. Themethods disclosed in these documents can be modified as described inU.S. Pat. No. 5,994,619, which is hereby incorporated by reference. U.S.Pat. No. 5,994,619 describes methods for producing novel cultured innercell mass (CICM) cells and cell lines, derived from pigs and cows, andtransgenic CICM cells into which heterologous DNA has been inserted.CICM transgenic cells can be used to produce cloned transgenic embryos,fetuses, and offspring. The '619 patent also describes methods ofproducing transgenic animals that are capable of transmitting theheterologous DNA to their progeny. In preferred embodiments of thecurrent invention, the non-human animals are mammals, particularly rats,sheep, pigs, goats, cattle, horses or chickens.

XENOMOUSE® mice produce an adult-like human repertoire of fully humanantibodies and generate antigen-specific human antibodies. In someembodiments, the XENOMOUSE® mice contain approximately 80% of the humanantibody V gene repertoire through introduction of megabase sized,germline configuration fragments of the human heavy chain loci and kappalight chain loci in yeast artificial chromosome (YAC). In otherembodiments, XENOMOUSE® mice further contain approximately all of thehuman lambda light chain locus. See Mendez et al., Nature Genetics15:146-156 (1997), Green and Jakobovits, J. Exp. Med. 188:483-495(1998), and WO 98/24893, the disclosures of which are herebyincorporated by reference.

In some embodiments, the non-human animal comprising humanimmunoglobulin genes are animals that have a human immunoglobulin“minilocus”. In the minilocus approach, an exogenous Ig locus ismimicked through the inclusion of individual genes from the Ig locus.Thus, one or more V_(H) genes, one or more D_(H) genes, one or moreJ_(H) genes, a mu constant domain, and a second constant domain(preferably a gamma constant domain) are formed into a construct forinsertion into an animal. This approach is described, inter alia, inU.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425,5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,591,669, 5,612,205,5,721,367, 5,789,215, and 5,643,763, hereby incorporated by reference.

In another aspect, the invention provides a method for making humanizedanti-myostatin antibodies. In some embodiments, non-human animals areimmunized with a myostatin antigen as described below under conditionsthat permit antibody production. Antibody-producing cells are isolatedfrom the animals, and nucleic acids encoding the heavy and light chainsof an anti-myostatin antibody of interest are isolated from the isolatedantibody-producing cells or from an immortalized cell line produced fromsuch cells. These nucleic acids are subsequently engineered usingtechniques known to those of skill in the art and as described furtherbelow to reduce the amount of non-human sequence, i.e., to humanize theantibody to reduce the immune response in humans.

In some embodiments, the myostatin antigen is isolated and/or purifiedmyostatin. In some embodiments, the myostatin antigen is humanmyostatin. However, because the C-terminal mature myostatin from humans,mice, rat, dog, quail, chicken, turkey, pig, horse, and monkeys isidentical and is not glycosylated in cells, myostatin from these speciesand other species having the same mature protein sequence also can beused as the immunogen. In other embodiments, the myostatin antigen is acell that expresses or overexpresses myostatin. In other embodiments,the myostatin antigen is a recombinant protein expressed from yeast,insect cells, bacteria such as E. Coli, or other resources byrecombinant technology.

Immunization of animals can be by any method known in the art. See,e.g., Harlow and Lane, Antibodies: A Laboratory Manual New York: ColdSpring Harbor Press, 1990. Methods for immunizing non-human animals suchas mice, rats, sheep, goats, pigs, cattle and horses are well known inthe art. See, e.g., Harlow and Lane, supra, and U.S. Pat. No. 5,994,619.In a preferred embodiment, the myostatin antigen is administered with anadjuvant to stimulate the immune response. Exemplary adjuvants includecomplete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) orISCOM (immunostimulating complexes). Such adjuvants may protect thepolypeptide from rapid dispersal by sequestering it in a local deposit,or they may contain substances that stimulate the host to secretefactors that are chemotactic for macrophages and other components of theimmune system. Preferably, if a polypeptide is being administered, theimmunization schedule will involve two or more administrations of thepolypeptide, spread out over several weeks. Example I exemplifies amethod for producing anti-myostatin monoclonal antibodies in XENOMOUSE®mice.

Production of Antibodies and Antibody-Producing Cell Lines

After immunization of an animal with a myostatin antigen, antibodiesand/or antibody-producing cells can be obtained from the animal. In someembodiments, anti-myostatin antibody-containing serum is obtained fromthe animal by bleeding or sacrificing the animal. The serum may be usedas it is obtained from the animal, an immunoglobulin fraction may beobtained from the serum, or the anti-myostatin antibodies may bepurified from the serum.

In some embodiments, antibody-producing cell lines are prepared fromcells isolated from the immunized animal. After immunization, the animalis sacrificed and lymph node and/or splenic B cells are immortalized byany means known in the art. Methods of immortalizing cells include, butare not limited to, transfecting them with oncogenes, infecting themwith an oncogenic virus and cultivating them under conditions thatselect for immortalized cells, subjecting them to carcinogenic ormutating compounds, fusing them with an immortalized cell, e.g., amyeloma cell, and inactivating a tumor suppressor gene. See, e.g.,Harlow and Lane, supra. If fusion with myeloma cells is used, themyeloma cells preferably do not secrete immunoglobulin polypeptides (anon-secretory cell line). Immortalized cells are screened usingmyostatin, or a portion thereof. In a preferred embodiment, the initialscreening is performed using an enzyme-linked immunoassay (ELISA) or aradioimmunoassay. An example of ELISA screening is provided in WO00/37504, incorporated herein by reference.

Anti-myostatin antibody-producing cells, e.g., hybridomas, are selected,cloned and further screened for desirable characteristics, includingrobust growth, high antibody production and desirable antibodycharacteristics, as discussed further below. Hybridomas can be expandedin vivo in syngeneic animals, in animals that lack an immune system,e.g., nude mice, or in cell culture in vitro. Methods of selecting,cloning and expanding hybridomas are well known to those of ordinaryskill in the art.

In a preferred embodiment, the immunized animal is a non-human animalthat expresses human immunoglobulin genes and the splenic B cells arefused to a myeloma cell line from the same species as the non-humananimal. In a more preferred embodiment, the immunized animal is aXENOMOUSE® mouse and the myeloma cell line is a non-secretory mousemyeloma. In an even more preferred embodiment, the myeloma cell line isP3-X63-Ag8.653 (American Type Culture Collection). See, e.g., Example I.

Thus, in one embodiment, the invention provides methods for producing acell line that produces a human monoclonal antibody or a fragmentthereof directed to myostatin comprising (a) immunizing a non-humantransgenic animal described herein with myostatin, a portion ofmyostatin or a cell or tissue expressing myostatin; (b) allowing thetransgenic animal to mount an immune response to myostatin; (c)isolating antibody-producing cells from transgenic animal; (d)immortalizing the antibody-producing cells; (e) creating individualmonoclonal populations of the immortalized antibody-producing cells; and(f) screening the immortalized antibody-producing cells to identify anantibody directed to myostatin.

In another aspect, the invention provides a cell line that produces ahuman anti-myostatin antibody. In some embodiments the cell line is ahybridoma cell line. In some embodiments, the hybridomas are mousehybridomas, as described above. In other embodiments, the hybridomas areproduced in a non-human, non-mouse species such as rats, sheep, pigs,goats, cattle or horses. In another embodiment, the hybridomas are humanhybridomas.

In another embodiment, a transgenic animal is immunized with a myostatinantigen, primary cells, e.g., spleen or peripheral blood B cells, areisolated from an immunized transgenic animal and individual cellsproducing antibodies specific for the desired antigen are identified.Polyadenylated mRNA from each individual cell is isolated and reversetranscription polymerase chain reaction (RT-PCR) is performed usingsense primers that anneal to variable domain sequences, e.g., degenerateprimers that recognize most or all of the FR1 regions of human heavy andlight chain variable domain genes and anti-sense primers that anneal toconstant or joining region sequences. cDNAs of the heavy and light chainvariable domains are then cloned and expressed in any suitable hostcell, e.g., a myeloma cell, as chimeric antibodies with respectiveimmunoglobulin constant regions, such as the heavy chain and K or λconstant domains. See Babcook, J. S. et al., Proc. Natl. Acad. Sci. USA93:7843-48, 1996, incorporated herein by reference. Anti myostatinantibodies may then be identified and isolated as described herein.

In another embodiment, phage display techniques can be used to providelibraries containing a repertoire of antibodies with varying affinitiesfor myostatin. For production of such repertoires, it is unnecessary toimmortalize the B cells from the immunized animal. Rather, the primary Bcells can be used directly as a source of mRNA. The mixture of cDNAsobtained from B cell, e.g., derived from spleens, is used to prepare anexpression library, for example, a phage display library transfectedinto E. coli. The resulting cells are tested for immunoreactivity tomyostatin. Techniques for the identification of high affinity humanantibodies from such libraries are described by Griffiths et al., EMBOJ., 13:3245-3260 (1994); Nissim et al., ibid, pp. 692-698 and byGriffiths et al., ibid, 12:725-734, which are incorporated by reference.Ultimately, clones from the library are identified that produce bindingaffinities of a desired magnitude for the antigen and the DNA encodingthe product responsible for such binding is recovered and manipulatedfor standard recombinant expression. Phage display libraries may also beconstructed using previously manipulated nucleotide sequences andscreened in a similar fashion. In general, the cDNAs encoding heavy andlight chains are independently supplied or linked to form Fv analogs forproduction in the phage library.

The phage library is then screened for the antibodies with the highestaffinities for myostatin and the genetic material recovered from theappropriate clone. Further rounds of screening can increase affinity ofthe original antibody isolated.

Nucleic Acids, Vectors, Host Cells, and Recombinant Methods of MakingAntibodies Nucleic Acids

The present invention also encompasses nucleic acid molecules encodinganti-myostatin antibodies or an antigen-binding fragments thereof. Insome embodiments, different nucleic acid molecules encode a heavy chainand a light chain of an anti-myostatin immunoglobulin. In otherembodiments, the same nucleic acid molecule encodes a heavy chain and alight chain of an anti-myostatin immunoglobulin.

In some embodiments, the nucleic acid molecule encoding the variabledomain of the light chain (V_(L)) utilizes a human A3, A30 or L2 VKgene, and a human JK1, JK3 or JK4 gene. In some embodiments the nucleicacid molecule utilizes a human A30, A3 or L2 VK gene and a human JK4gene. In other embodiments, the nucleic acid molecule utilizes a humanA30, A3 or L2 gene and a human JK1 gene. In some embodiments, thenucleic acid molecule encoding the light chain encodes an amino acidsequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions fromthe germline amino acid sequence(s). In some embodiments, the nucleicacid molecule comprises a nucleotide sequence that encodes a V_(L) aminoacid sequence comprising 1, 2, 3, 4 or 5 conservative amino acidsubstitutions and/or 1, 2, or 3 non-conservative substitutions comparedto germline V_(K) and J_(K) sequences. Substitutions may be in the CDRregions, the framework regions, or in the constant domain.

In some embodiments, the nucleic acid molecule encodes a V_(L) aminoacid sequence comprising one or more mutations compared to the germlinesequence that are identical to the mutations from germline found in theV_(L) of any one of antibodies 1_(—)116_(—)1, 1_(—)136_(—)3,1_(—)257_(—)1, 1_(—)46_(—)1, 2_(—)112_(—), 1_(—)314_(—)1, 1_(—)66_(—)1,2_(—)43_(—)1, 2_(—)177_(—)1, 1_(—)132_(—)1, 1_(—)268_(—)1,1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I; 1_(—)314_(—)1H-T92A;1_(—)46_(—)1H-L81M; 2_(—)112_(—)1H-I12V; 2_(—)112_(—)1L-F58I;2_(—)112_(—)1L-I85V; 2_(—)112_(—)1H-L81M, L-F58I; 2_(—)112_(—)1H-L81M,L-I85V; or 2_(—)112_(—)1H-L81M, L-F58I, I85V.

In some embodiments, the nucleic acid molecule encodes at least threeamino acid substitutions compared to the germline sequence found in theV_(L) of any one of the antibodies 1_(—)116_(—)1, 1_(—)136_(—)3,1_(—)257_(—)1, 1_(—)46_(—)1, 2_(—)112_(—), 1_(—)314_(—)1, 1_(—)66_(—)1,2_(—)43_(—)1, 2_(—)177_(—)1, 1_(—)132_(—)1, 1_(—)268_(—)1,1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I; 1_(—)314_(—)1H-T92A;1_(—)46_(—)1H-L81M; 2_(—)112_(—)1H-I12V; 2_(—)112_(—)1L-F58I;2_(—)112_(—)1L-I85V; 2_(—)112_(—)1H-L81M, L-F58I; 2_(—)112_(—)1H-L81M,L-I85V; or 2_(—)112_(—)1H-L81M, L-F58I, I85V.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence that encodes the V_(L) amino acid sequence of monoclonalantibody 1_(—)116_(—)1 (SEQ ID NO: 4); 1_(—)136_(—)3 (SEQ ID NO: 12);1_(—)257_(—)1 (SEQ ID NO: 16); 1_(—)46_(—)1 (SEQ ID NO: 24);2_(—)112_(—)1 (SEQ ID NO: 28); 1_(—)314_(—)1 (SEQ ID NO: 20-_);1_(—)66_(—)1 (SEQ ID NO: 36); 2_(—)43_(—)1 (SEQ ID NO: 28);2_(—)177_(—)1 (SEQ ID NO: 40); 1_(—)132_(—)1 (SEQ ID NO: 8); or1_(—)268_(—)1 (SEQ ID NO: 32) or a variant or portion thereof. In someembodiments, the nucleic acid encodes an amino acid sequence comprisingthe light chain CDRs of one of said above-listed antibodies. In someembodiments, said portion is a contiguous portion comprising CDR1-CDR3.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence that encodes the amino acid sequence of one of SEQ ID NOs: 4,8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 47, 51, 55, 59, 63, 67, 71, 75,79, 83 or 87. In some embodiments, the nucleic acid molecule comprisesthe nucleotide sequence of SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35,39, 43, 48, 52, 56, 60, 68, 72, 80, 64, 76, 84 or 88 or a portionthereof. In some embodiments, the nucleic acid encodes the amino acidsequence of the light chain of one, two or all three CDRs of saidantibody. In some embodiments, said portion encodes a contiguous regionfrom CDR1-CDR3 of the light chain of an anti-myostatin antibody.

In some embodiments, the nucleic acid molecule encodes a V_(L) aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or99% identical to the V_(L) amino acid sequence of any one of antibodies1_(—)116_(—)1, 1_(—)136_(—)3, 1_(—)257_(—)1, 1_(—)46_(—)1,2_(—)112_(—)1, 1_(—)314_(—)1, 1_(—)66_(—)1, 2_(—)43_(—)1, 2_(—)177_(—)1,1_(—)132_(—)1, 1_(—)268_(—)1, 1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I;1 _(—)314_(—)1H-T92A; 1_(—)46_(—)1H-L81M; 2_(—)112_(—)1H-I12V;2_(—)112_(—)1L-F58I; 2_(—)112_(—)1L-I85V; or 2_(—)112_(—)1H-L81M,L-F58I; 2_(—)112_(—)1H-L81M, L-I85V; or 2_(—)112_(—)1H-L81M, L-F58I,I85V, or to the amino acid sequence of the V_(L) region of any one ofSEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 47, 51, 55, 59,63, 67, 71, 75, 79, 83 or 87. Nucleic acid molecules of the inventioninclude nucleic acids that hybridize under highly stringent conditions,such as those described above, or that are at least 70%, 75%, 80%, 85%,90%, 95%, 97%, 98% or 99% identical to a nucleic acid encoding the aminoacid sequence the V_(L) region of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28,32, 36, 40, 44, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83 or 87 or to anucleic acid comprising the V_(L) region nucleotide sequence of SEQ IDNOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 48, 52, 56, 60, 68, 72,80, 64, 76, 84 or 88

In other preferred embodiments, the nucleic acid molecule encodes thevariable domain of a heavy chain (V_(H)) that utilizes a human 1-02,3-21 or 3-23 V_(H) gene sequence or a sequence derived therefrom. Invarious embodiments, the nucleic acid molecule utilizes a human V_(H)1-02 gene, a D4-23 gene and a human J_(H)6b gene; a human V_(H) 3-21gene, a human D3-16 or D5-12 gene and a human J_(H)6b gene; a humanV_(H) 3-21 gene, a human D1-26 or D5-5 gene and a human J_(H)4b gene; ahuman V_(H) 3-23 gene, a human D1-7 gene and a human J_(H)3b gene.

In some embodiments, the nucleic acid molecule encodes an amino acidsequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 mutationscompared to the germline amino acid sequence of the human V, D or Jgenes. In some embodiments, said mutations are in the V_(H) region. Insome embodiments, said mutations are in the CDR regions.

In some embodiments, the nucleic acid molecule encodes a V_(H) sequencecomprising one or more amino acid mutations compared to the germlineV_(H) sequence that are identical to amino acid mutations found in theV_(H) of monoclonal antibody 1_(—)116_(—)1, 1_(—)136_(—)3,1_(—)257_(—)1, 1_(—)46_(—)1, 2_(—)112_(—), 1_(—)314_(—)1, 1_(—)66_(—)1,2_(—)43_(—)1, 2_(—)177_(—)1, 1_(—)132_(—)1, 1_(—)268_(—)1,1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I; 1_(—)314_(—)1H-T92A;1_(—)46_(—)1H-L81M; 2_(—)112_(—)1H-I12V; 2_(—)112_(—)1L-F58I;2_(—)112_(—)1L-I85V; 2_(—)112_(—)1H-L81M, L-F58I; 2_(—)112_(—)1H-L81M,L-I85V; or 2_(—)112_(—)1H-L81M, L-F58I, I85V. In some embodiments, thenucleic acid encodes at least three amino acid mutations compared to thegermline sequences that are identical to at least three amino acidmutations found in one of the above-listed monoclonal antibodies.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence that encodes at least a portion of the V_(H) amino acidsequence of a monoclonal antibody selected from 1_(—)116_(—)1 (SEQ IDNO: 2); 1_(—)136_(—)3 (SEQ ID NO: 10); 1_(—)257_(—)1 (SEQ ID NO: 14);1_(—)46_(—)1 (SEQ ID NO: 22); 2_(—)112_(—)1 (SEQ ID NO: 26);1_(—)314_(—)1 (SEQ ID NO: 18); 1_(—)66_(—)1 (SEQ ID NO: 34);2_(—)43_(—)1 (SEQ ID NO: 42); 2_(—)177_(—)1 (SEQ ID NO: 38);1_(—)132_(—)1 (SEQ ID NO: 6); or 1_(—)268_(—)1 (SEQ ID NO: 30), avariant thereof, or said sequence having conservative amino acidmutations and/or a total of three or fewer non-conservative amino acidsubstitutions. In various embodiments the sequence encodes one or moreCDR regions, preferably a CDR3 region, all three CDR regions, acontiguous portion including CDR1-CDR3, or the entire V_(H) region, ofan above-listed anti-myostatin antibody.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence that encodes the amino acid sequence of one of SEQ ID NOs: 2,6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 45, 49, 53, 57, 61, 65, 69, 73,77, 81 or 85. In various preferred embodiments, the nucleic acidmolecule comprises at least a portion of the nucleotide sequence of SEQID NOS: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 46, 50, 54, 58, 62, 66,70, 74, 78, 82 or 86. In some embodiments, said portion encodes theV_(H) region, a CDR3 region, all three CDR regions, or a contiguousregion including CDR1-CDR3.

In some embodiments, the nucleic acid molecule encodes a V_(H) aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or99% identical to the V_(H) amino acid sequence in any one of SEQ ID NOS:2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 45, 49, 53, 57, 61, 65, 69,73, 77, 81 or 85. Nucleic acid molecules of the invention includenucleic acids that hybridize under highly stringent conditions, such asthose described above, or that are at least 70%, 75%, 80%, 85%, 90%,95%, 97%, 98% or 99% identical to a nucleic acid encoding the amino acidsequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38 or 42 or toa V_(H) region thereof, or to a nucleic acid comprising the nucleotidesequence of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37 or 41 or thenucleotide sequence that encodes a V_(H) region thereof.

In another embodiment, the nucleic acid encodes a full-length heavychain of an antibody selected from 1_(—)116_(—)1, 1_(—)136_(—)3,1_(—)257_(—)1, 1_(—)46_(—)1, 2_(—)112_(—), 1_(—)314_(—)1, 1_(—)66_(—)1,2_(—)43_(—)1, 2_(—)177_(—)1, 1_(—)132_(—)1, 1_(—)268_(—)1,1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I; 1_(—)314_(—)1H-T92A;1_(—)46_(—)1H-L81M; 2_(—)112_(—)1H-I12V; 2_(—)112_(—)1L-F58I;2_(—)112_(—)1L-I85V; 2_(—)112_(—)1H-L81M, L-F58I; 2 _(—)112_(—)1H-L81M,L-I85V; or 2_(—)112_(—)1H-L81M, L-F58I, I85V, or a heavy chaincomprising the amino acid sequence of SEQ ID NOs: 2, 6, 10, 14, 18, 22,26, 30, 34, 38 or 42. Further, the nucleic acid may comprise thenucleotide sequence of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37or 41.

A nucleic acid molecule encoding the heavy or light chain of ananti-myostatin antibody or portions thereof can be isolated from anysource that produces such antibody. In various embodiments, the nucleicacid molecules are isolated from a B cell that expresses ananti-myostatin antibody isolated from an animal immunized with myostatinor from an immortalized cell derived from such a B cell. Methods ofisolating nucleic acids encoding an antibody are well-known in the art.See, e.g., Sambrook et al. mRNA may be isolated and used to produce cDNAfor use in the polymerase chain reaction (PCR) or cDNA cloning ofantibody genes. In a preferred embodiment, the nucleic acid molecule isisolated from a hybridoma that has as one of its fusion partners a cellfrom a non-human transgenic animal, said cell producing a humanimmunoglobulin. In an even more preferred embodiment, the cell producinghuman immunoglobulin is isolated from a XENOMOUSE® animal. In anotherembodiment, the cell producing the human immunoglobulin is isolated froma non-human, non-mouse transgenic animal, as described above. In anotherembodiment, the nucleic acid is isolated from a non-human,non-transgenic animal. The nucleic acid molecules isolated from anon-human, non-transgenic animal may be used, e.g., for humanizedantibodies that comprise one or more amino acid sequences from a humananti-myostatin antibody of the present invention.

In some embodiments, a nucleic acid encoding a heavy chain of ananti-myostatin antibody of the invention can comprise a nucleotidesequence encoding a V_(H) domain of the invention joined in-frame to anucleotide sequence encoding a heavy chain constant domain from anysource. Similarly, a nucleic acid molecule encoding a light chain of ananti-myostatin antibody of the invention can comprise a nucleotidesequence encoding a V_(L) domain of the invention joined in-frame to anucleotide sequence encoding a light chain constant domain from anysource.

In a further aspect of the invention, nucleic acid molecules encodingthe variable domain of the heavy (V_(H)) and/or light (V_(L)) chains are“converted” to full-length antibody genes. In one embodiment, nucleicacid molecules encoding the V_(H) or V_(L) domains are converted tofull-length antibody genes by insertion into an expression vectoralready encoding heavy chain constant (C_(H)) or light chain constant(C_(L)) domains, respectively, such that the V_(H) segment isoperatively linked to the C_(H) segment(s) within the vector, and/or theV_(L) segment is operatively linked to the C_(L) segment within thevector. In another embodiment, nucleic acid molecules encoding the V_(H)and/or V_(L) domains are converted into full-length antibody genes bylinking, e.g., ligating, a nucleic acid molecule encoding a V_(H) and/orV_(L) domains to a nucleic acid molecule encoding a C_(H) and/or C_(L)domain using standard molecular biological techniques. Nucleic acidsequences of human heavy and light chain immunoglobulin constant domaingenes are known in the art. See, e.g., Kabat et al. Sequences ofProteins of Immunological Interest, 5th Ed., NIH Publ. No. 91-3242,1991. Nucleic acid molecules encoding the full-length heavy and/or lightchains may then be expressed from a cell into which they have beenintroduced and the anti-myostatin antibody isolated.

The nucleic acid molecules may be used to recombinantly express largequantities of anti-myostatin antibodies. The nucleic acid molecules alsomay be used to produce chimeric antibodies, bispecific antibodies,single chain antibodies, immunoadhesins, diabodies, mutated antibodiesand antibody derivatives, as described further below. If the nucleicacid molecules are derived from a non-human, non-transgenic animal, thenucleic acid molecules may be used for antibody humanization, also asdescribed below.

In another embodiment, a nucleic acid molecule of the invention is usedas a probe or PCR primer for a specific antibody sequence. For instance,the nucleic acid can be used as a probe in diagnostic methods or as aPCR primer to amplify regions of DNA that could be used, inter alia, toisolate additional nucleic acid molecules encoding variable domains ofanti-myostatin antibodies. In some embodiments, the nucleic acidmolecules are oligonucleotides. In some embodiments, theoligonucleotides are from highly variable domains of the heavy and lightchains of the antibody of interest. In some embodiments, theoligonucleotides encode all or a part of one or more of the CDRs ofantibodies 1_(—)116_(—)1, 1_(—)136_(—)3, 1_(—)257_(—)1, 1_(—)46_(—)1,2_(—)112_(—), 1_(—)314_(—)1, 1_(—)66_(—)1, 2_(—)43_(—)1, 2_(—)177_(—)1,1_(—)132_(—)1, 1_(—)268_(—)1, 1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I;1_(—)314_(—)1H-T92A; 1_(—)46_(—)1H-L81M; 2_(—)112_(—)1H-I12V;2_(—)112_(—)1L-F58I; 2_(—)112_(—)1L-I85V; 2_(—)112_(—)1H-L81M, L-F581;or 2_(—)112_(—)1H-L81M, L-I85V; or 2_(—)112_(—)1H-L81M, L-F58I, I85V orvariants thereof as described herein.

Vectors

The invention provides vectors comprising nucleic acid molecules thatencode the heavy chain of an anti-myostatin antibody of the invention oran antigen-binding portion thereof. The invention also provides vectorscomprising nucleic acid molecules that encode the light chain of suchantibodies or antigen-binding portion thereof. The invention furtherprovides vectors comprising nucleic acid molecules encoding fusionproteins, modified antibodies, antibody fragments, and probes thereof.

In some embodiments, the anti-myostatin antibodies of the invention orantigen-binding portions are expressed by inserting DNAs encodingpartial or full-length light and heavy chains, obtained as describedabove, into expression vectors such that the genes are operativelylinked to necessary expression control sequences such as transcriptionaland translational control sequences. Expression vectors includeplasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV),plant viruses such as cauliflower mosaic virus, tobacco mosaic virus,cosmids, YACs, EBV derived episomes, and the like. The antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevectors. In a preferred embodiment, both genes are inserted into thesame expression vector. The antibody genes are inserted into theexpression vector by standard methods (e.g., ligation of complementaryrestriction sites on the antibody gene fragment and vector, or blunt endligation if no restriction sites are present).

A convenient vector is one that encodes a functionally complete humanC_(H) or C_(L) immunoglobulin sequence, with appropriate restrictionsites engineered so that any V_(H) or V_(L) sequence can easily beinserted and expressed, as described above. In such vectors, splicingusually occurs between the splice donor site in the inserted J regionand the splice acceptor site preceding the human C domain, and also atthe splice regions that occur within the human C_(H) exons.Polyadenylation and transcription termination occur at nativechromosomal sites downstream of the coding regions. The recombinantexpression vector also can encode a signal peptide that facilitatessecretion of the antibody chain from a host cell. The antibody chaingene may be cloned into the vector such that the signal peptide islinked in-frame to the amino terminus of the immunoglobulin chain. Thesignal peptide can be an immunoglobulin signal peptide or a heterologoussignal peptide (i.e., a signal peptide from a non-immunoglobulinprotein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. It will beappreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Preferred regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from retroviral LTRs, cytomegalovirus(CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (suchas the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus majorlate promoter (AdMLP)), polyoma and strong mammalian promoters such asnative immunoglobulin and actin promoters. For further description ofviral regulatory elements, and sequences thereof, see e.g., U.S. Pat.No. 5,168,062, U.S. Pat. No. 4,510,245 and U.S. Pat. No. 4,968,615.Methods for expressing antibodies in plants, including a description ofpromoters and vectors, as well as transformation of plants is known inthe art. See, e.g., U.S. Pat. No. 6,517,529, incorporated herein byreference. Methods of expressing polypeptides in bacterial cells orfungal cells, e.g., yeast cells, are also well known in the art.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, incorporated herein by reference).For example, typically the selectable marker gene confers resistance todrugs, such as G418, hygromycin or methotrexate, on a host cell intowhich the vector has been introduced. Preferred selectable marker genesinclude the dihydrofolate reductase (DHFR) gene (for use in dhfr-hostcells with methotrexate selection/amplification), the neo gene (for G418selection), and the glutamate synthetase gene.

Non-Hybridoma Host Cells and Methods of Recombinantly Producing Protein

Nucleic acid molecules encoding anti-myostatin antibodies and vectorscomprising these nucleic acid molecules can be used for transfection ofa suitable mammalian, plant, bacterial or yeast host cell.Transformation can be by any known method for introducingpolynucleotides into a host cell. Methods for introduction ofheterologous polynucleotides into mammalian cells are well known in theart and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene-mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei. In addition, nucleicacid molecules may be introduced into mammalian cells by viral vectors.Methods of transforming cells are well known in the art. See, e.g., U.S.Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, incorporatedherein by reference). Methods of transforming plant cells are well knownin the art, including, e.g., Agrobacterium-mediated transformation,biolistic transformation, direct injection, electroporation and viraltransformation. Methods of transforming bacterial and yeast cells arealso well known in the art.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC). These include, inter alia,Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells,NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, Africangreen monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), A549 cells, and a number of other cell lines. Cell linesof particular preference are selected through determining which celllines have high expression levels.

Cell lines other than those of mammalian origin can also be used. Theseinclude insect cell lines (such as Sf9 or Sf21 cells), plant cells(including those from Nicotiana, Arabidopsis, duckweed, corn, wheat,potato, etc.), bacterial cells (including E. coli and Streptomyces) andyeast cells (including Schizosaccharomyces pombe, Saccharomycescerevisiae and Pichia pastoris).

When recombinant expression vectors encoding antibody genes areintroduced into host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or, more preferably, secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

Expression of antibodies of the invention from production cell lines canbe enhanced using a number of known techniques. For example, theglutamine synthetase gene expression system (the GS system) is a commonapproach for enhancing expression under certain conditions. The GSsystem is discussed in whole or part in connection with European PatentNos. 0 216 846, 0 256 055, 0 323 997 and 0 338 841.

It is likely that antibodies expressed by different cell lines or intransgenic animals will have different glycosylation from each other.However, all antibodies encoded by the nucleic acid molecules providedherein, or comprising the amino acid sequences provided herein are partof the instant invention, regardless of the glycosylation of theantibodies.

Transgenic Animals and Plants

Anti-myostatin antibodies of the invention also can be producedtransgenically through the generation of a mammal or plant that istransgenic for the immunoglobulin heavy and light chain sequences ofinterest and production of the antibody in a recoverable form therefrom.In connection with the transgenic production in mammals, anti-myostatinantibodies can be produced in, and recovered from, the milk of goats,cows, or other mammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687,5,750,172, and 5,741,957, incorporated herein by reference. In someembodiments, non-human transgenic animals that comprise humanimmunoglobulin loci are immunized with myostatin or an immunogenicportion thereof, as described above. Methods for making antibodies inplants are described, e.g., in U.S. Pat. Nos. 6,046,037 and 5,959,177,incorporated herein by reference.

In some embodiments, non-human transgenic animals or plants are producedby introducing one or more nucleic acid molecules encoding ananti-myostatin antibody of the invention into the animal or plant bystandard transgenic techniques. See Hogan and U.S. Pat. No. 6,417,429,supra. The transgenic cells used for making the transgenic animal can beembryonic stem cells or somatic cells or a fertilized egg. Thetransgenic non-human organisms can be chimeric, nonchimericheterozygotes, and nonchimeric homozygotes. See, e.g., Hogan et al.,Manipulating the Mouse Embryo: A Laboratory Manual 2^(nd) ed., ColdSpring Harbor Press (1999); Jackson et al., Mouse Genetics andTransgenics: A Practical Approach, Oxford University Press (2000); andPinkert Transgenic Animal Technology: A Laboratory Handbook, AcademicPress (1999), all incorporated herein by reference. In some embodiments,the transgenic non-human animals have a targeted disruption andreplacement by a targeting construct that encodes a heavy chain and/or alight chain of interest. In a preferred embodiment, the transgenicanimals comprise and express nucleic acid molecules encoding heavy andlight chains that specifically bind to myostatin, preferably humanmyostatin. In some embodiments, the transgenic animals comprise nucleicacid molecules encoding a modified antibody such as a single-chainantibody, a chimeric antibody or a humanized antibody. Theanti-myostatin antibodies may be made in any transgenic animal. In apreferred embodiment, the non-human animals are mice, rats, sheep, pigs,goats, cattle or horses. The non-human transgenic animal expresses saidencoded polypeptides in blood, milk, urine, saliva, tears, mucus andother bodily fluids.

Phage Display Libraries

The invention provides a method for producing an anti-myostatin antibodyor antigen-binding portion thereof comprising the steps of synthesizinga library of human antibodies on phage, screening the library withmyostatin or an antibody-binding portion thereof, isolating phage thatbind myostatin, and obtaining the antibody from the phage. By way ofexample, one method for preparing the library of antibodies for use inphage display techniques comprises the steps of immunizing a non-humananimal comprising human immunoglobulin loci with myostatin or anantigenic portion thereof to create an immune response, extractingantibody-producing cells from the immunized animal; isolating RNAencoding heavy and light chains of antibodies of the invention from theextracted cells, reverse transcribing the RNA to produce cDNA,amplifying the cDNA using primers, and inserting the cDNA into a phagedisplay vector such that antibodies are expressed on the phage.Recombinant anti-myostatin antibodies of the invention may be obtainedin this way.

Recombinant human anti-myostatin antibodies of the invention can beisolated by screening a recombinant combinatorial antibody library.Preferably the library is a scFv phage display library, generated usinghuman V_(L) and V_(H) cDNAs prepared from mRNA isolated from B cells.Methods for preparing and screening such libraries are known in the art.Kits for generating phage display libraries are commercially available(e.g., the Pharmacia Recombinant Phage Antibody System, catalog no.27-9400-01; and the Stratagene SURFZAP phage display kit, catalog no.240612). There also are other methods and reagents that can be used ingenerating and screening antibody display libraries (see, e.g., U.S.Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619, WO 91/17271, WO92/20791, WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs etal., Bio/Technology 9:1370-1372 (1991); Hay et al., Hum. Antibod.Hybridomas 3:81-85 (1992); Huse et al., Science 246:1275-1281 (1989);McCafferty et al., Nature 348:552-554 (1990); Griffiths et al., EMBO J.12:725-734 (1993); Hawkins et al., J. Mol. Biol. 226:889-896 (1992);Clackson et al., Nature 352:624-628 (1991); Gram et al., Proc. Natl.Acad. Sci. USA 89:3576-3580 (1992); Garrad et al., Bio/Technology9:1373-1377 (1991); Hoogen boom et al., Nuc. Acid Res. 19:4133-4137(1991); and Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982(1991), all incorporated herein by reference.

In one embodiment, to isolate and produce human anti-myostatinantibodies with the desired characteristics, a human anti-myostatinantibody as described herein is first used to select human heavy andlight chain sequences having similar binding activity toward myostatin,using the epitope imprinting methods described in PCT Publication No. WO93/06213, incorporated herein by reference. The antibody libraries usedin this method are preferably scFv libraries prepared and screened asdescribed in PCT Publication No. WO 92/01047, McCafferty et al., Nature348:552-554 (1990); and Griffiths et al., EMBO J. 12:725-734 (1993), allincorporated herein by reference. The scFv antibody libraries preferablyare screened using human myostatin as the antigen.

Once initial human V_(L) and V_(H) domains are selected, “mix and match”experiments are performed, in which different pairs of the initiallyselected V_(L) and V_(H) segments are screened for myostatin binding toselect preferred V_(L)/V_(H) pair combinations. Additionally, to furtherimprove the quality of the antibody, the V_(L) and V_(H) segments of thepreferred V_(L)/V_(H) pair(s) can be randomly mutated, preferably withinthe CDR3 region of V_(H) and/or V_(L), in a process analogous to the invivo somatic mutation process responsible for affinity maturation ofantibodies during a natural immune response. This in vitro affinitymaturation can be accomplished by amplifying V_(H) and V_(L) domainsusing PCR primers complimentary to the V_(H) CDR3 or V_(L) CDR3,respectively, which primers have been “spiked” with a random mixture ofthe four nucleotide bases at certain positions such that the resultantPCR products encode V_(H) and V_(L) segments into which random mutationshave been introduced into the V_(H) and/or V_(L) CDR3 regions. Theserandomly mutated V_(H) and V_(L) segments can be re-screened for bindingto myostatin.

Following screening and isolation of an anti-myostatin antibody of theinvention from a recombinant immunoglobulin display library, nucleicacids encoding the selected antibody can be recovered from the displaypackage (e.g., from the phage genome) and subcloned into otherexpression vectors by standard recombinant DNA techniques. If desired,the nucleic acid can further be manipulated to create other antibodyforms of the invention, as described below. To express a recombinanthuman antibody isolated by screening of a combinatorial library, the DNAencoding the antibody is cloned into a recombinant expression vector andintroduced into a mammalian host cells, as described above.

Class Switching

Another aspect of the invention provides a method for converting theclass or subclass of an anti-myostatin antibody to another class orsubclass. In some embodiments, a nucleic acid molecule encoding a V_(L)or V_(H) that does not include sequences encoding C_(L) or C_(H) isisolated using methods well-known in the art. The nucleic acid moleculethen is operatively linked to a nucleic acid sequence encoding a C_(L)or C_(H) from a desired immunoglobulin class or subclass. This can beachieved using a vector or nucleic acid molecule that comprises a C_(L)or C_(H) chain, as described above. For example, an anti-myostatinantibody that was originally IgM can be class switched to an IgG.Further, the class switching may be used to convert one IgG subclass toanother, e.g., from IgG1 to IgG2. Another method for producing anantibody of the invention comprising a desired isotype comprises thesteps of isolating a nucleic acid encoding a heavy chain of ananti-myostatin antibody and a nucleic acid encoding a light chain of ananti-myostatin antibody, isolating the sequence encoding the V_(H)region, ligating the V_(H) sequence to a sequence encoding a heavy chainconstant domain of the desired isotype, expressing the light chain geneand the heavy chain construct in a cell, and collecting theanti-myostatin antibody with the desired isotype.

Deimmunized Antibodies

In another aspect of the invention, the antibody may be deimmunized toreduce its immunogenicity using the techniques described in, e.g., PCTPublication Nos. WO98/52976 and WO00/34317 (incorporated herein byreference).

Mutated Antibodies

In another embodiment, the nucleic acid molecules, vectors and hostcells may be used to make mutated anti-myostatin antibodies. Theantibodies may be mutated in the variable domains of the heavy and/orlight chains, e.g., to alter a binding property of the antibody. Forexample, a mutation may be made in one or more of the CDR regions toincrease or decrease the K_(D) of the antibody for myostatin, toincrease or decrease k_(off), or to alter the binding specificity of theantibody. Techniques in site-directed mutagenesis are well-known in theart. See, e.g., Sambrook et al. and Ausubel et al., supra. In anotherembodiment, one or more mutations are made at an amino acid residue thatis known to be changed compared to the germline in monoclonal antibody1_(—)116_(—)1, 1_(—)136_(—)3, 1_(—)257_(—)1, 1_(—)46_(—)1, 2_(—)112_(—),1_(—)314_(—)1, 1_(—)66_(—)1, 2_(—)43_(—)1, 2_(—)177_(—)1, 1_(—)132_(—)1,1_(—)268_(—)1, 1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I;1_(—)314_(—)1H-T92A; 1_(—)46_(—)1H-L81M; 2_(—)112_(—)1H-I12V;2_(—)112_(—)1L-F58I; 2_(—)112_(—)1L-I85V; 2_(—)112_(—)1H-L81M, L-F58I;2_(—)112_(—)1H-L81M, L-I85V; or 2_(—)112_(—)1H-L81M, L-F58I, I85V. Themutations may be made in a CDR region or framework region of a variabledomain, or in a constant domain. In a preferred embodiment, themutations are made in a variable domain. In some embodiments, one ormore mutations are made at an amino acid residue that is known to bechanged compared to the germline in a CDR region or framework region ofa variable domain of an amino acid sequence selected from SEQ ID NO: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,77, 79, 81, 83, 85 or 87 or whose nucleic acid sequence is presented inSEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86 or 88.

In another embodiment, the framework region is mutated so that theresulting framework region(s) have the amino acid sequence of thecorresponding germline gene. A mutation may be made in a frameworkregion or constant domain to increase the half-life of theanti-myostatin antibody. See, e.g., PCT Publication No. WO 00/09560,incorporated herein by reference. A mutation in a framework region orconstant domain also can be made to alter the immunogenicity of theantibody, to provide a site for covalent or non-covalent binding toanother molecule, or to alter such properties as complement fixation,FcR binding and antibody-dependent cell-mediated cytotoxicity (ADCC).According to the invention, a single antibody may have mutations in anyone or more of the CDRs or framework regions of the variable domain orin the constant domain.

In some embodiments, there are from 1 to 8, including any number inbetween, amino acid mutations in either the V_(H) or V_(L) domains ofthe mutated anti-myostatin antibody compared to the anti-myostatinantibody prior to mutation. In any of the above, the mutations may occurin one or more CDR regions. Further, any of the mutations can beconservative amino acid substitutions. In some embodiments, there are nomore than 5, 4, 3, 2, or 1 amino acid changes in the constant domains.

Modified Antibodies

In another embodiment, a fusion antibody or immunoadhesin may be madethat comprises all or a portion of an anti-myostatin antibody of theinvention linked to another polypeptide. In a preferred embodiment, onlythe variable domains of the anti-myostatin antibody are linked to thepolypeptide. In another preferred embodiment, the V_(H) domain of ananti-myostatin antibody is linked to a first polypeptide, while theV_(L) domain of an anti-myostatin antibody is linked to a secondpolypeptide that associates with the first polypeptide in a manner suchthat the V_(H) and V_(L) domains can interact with one another to forman antigen binding site. In another preferred embodiment, the V_(H)domain is separated from the V_(L) domain by a linker such that theV_(H) and V_(L) domains can interact with one another (see below underSingle Chain Antibodies). The V_(H)-linker-V_(L) antibody is then linkedto the polypeptide of interest. In addition, fusion antibodies can becreated in which two (or more) single-chain antibodies are linked to oneanother. This is useful if one wants to create a divalent or polyvalentantibody on a single polypeptide chain, or if one wants to create abispecific antibody.

To create a single chain antibody, (scFv) the V_(H)- and V_(L)-encodingDNA fragments are operatively linked to another fragment encoding aflexible linker, e.g., encoding the amino acid sequence (Gly₄-Ser)₃,(SEQ ID NO:122) such that the V_(H) and V_(L) sequences can be expressedas a contiguous single-chain protein, with the V_(L) and V_(H) domainsjoined by the flexible linker. See, e.g., Bird et al., Science242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA85:5879-5883 (1988); McCafferty et al., Nature 348:552-554 (1990). Thesingle chain antibody may be monovalent, if only a single V_(H) andV_(L) are used, bivalent, if two V_(H) and V_(L) are used, orpolyvalent, if more than two V_(H) and V_(L) are used. Bispecific orpolyvalent antibodies may be generated that bind specifically tomyostatin and to another molecule.

In other embodiments, other modified antibodies may be prepared usinganti-myostatin antibody encoding nucleic acid molecules. For instance,“Kappa bodies” (III et al., Protein Eng. 10: 949-57 (1997)),“Minibodies” (Martin et al., EMBO J. 13: 5303-9 (1994)), “Diabodies”(Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)), or“Janusins” (Traunecker et al., EMBO J. 10:3655-3659 (1991) andTraunecker et al., Int. J. Cancer (Suppl.) 7:51-52 (1992)) may beprepared using standard molecular biological techniques following theteachings of the specification.

Bispecific antibodies or antigen-binding fragments can be produced by avariety of methods including fusion of hybridomas or linking of Fab′fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990), Kostelny et al., J. Immunol. 148:1547-1553 (1992). Inaddition, bispecific antibodies may be formed as “diabodies” or“Janusins.” In some embodiments, the bispecific antibody binds to twodifferent epitopes of myostatin. In some embodiments, the bispecificantibody has a first heavy chain and a first light chain from monoclonalantibody 1_(—)116_(—)1, 1_(—)136_(—)3, 1_(—)257_(—)1, 1_(—)46_(—)1,2_(—)112_(—), 1_(—)314_(—)1, 1_(—)66_(—)1, 2_(—)43_(—)1, 2_(—)177_(—)1,1_(—)132_(—)1, 1_(—)268_(—)1, 1_(—)116_(—)1L-Q45K; 1_(—)257_(—)1L-L21I;1_(—)314_(—)1H-T92A; 1_(—)46_(—)1H-L81M; 2_(—)112_(—)1H-I12V;2_(—)112_(—)1L-F58I; 2_(—)112_(—)1L-I85V; 2_(—)112_(—)1H-L81M, L-F58I;2_(—)112_(—)1H-L81M, L-I85V; or 2_(—)112_(—)1H-L81M, L-F58I, I85V and anadditional antibody heavy chain and light chain. In some embodiments,the additional light chain and heavy chain also are from one of theabove-identified monoclonal antibodies, but are different from the firstheavy and light chains.

In some embodiments, the modified antibodies described above areprepared using one or more of the variable domains or CDR regions from ahuman anti-myostatin monoclonal antibody provided herein.

Derivatized and Labeled Antibodies

An anti-myostatin antibody or antigen-binding portion of the inventioncan be derivatized or linked to another molecule (e.g., another peptideor protein). In general, the antibodies or portion thereof arederivatized such that the myostatin binding is not affected adversely bythe derivatization or labeling. Accordingly, the antibodies and antibodyportions of the invention are intended to include both intact andmodified forms of the human anti-myostatin antibodies described herein.For example, an antibody or antibody portion of the invention can befunctionally linked (by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other molecular entities, suchas another antibody (e.g., a bispecific antibody or a diabody), adetection agent, a pharmaceutical agent, and/or a protein or peptidethat can mediate association of the antibody or antibody portion withanother molecule (such as a streptavidin core region or a polyhistidinetag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Another type of derivatized antibody is a labeled antibody. Usefuldetection agents with which an antibody or antigen-binding portion ofthe invention may be derivatized include fluorescent compounds,including fluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. An antibody can also be labeled with enzymesthat are useful for detection, such as horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase andthe like. When an antibody is labeled with a detectable enzyme, it isdetected by adding additional reagents that the enzyme uses to produce areaction product that can be discerned. For example, when the agenthorseradish peroxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody can also be labeled with biotin, and detectedthrough indirect measurement of avidin or streptavidin binding. Anantibody can also be labeled with a predetermined polypeptide epitoperecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some embodiments, labels are attached by spacer arms ofvarious lengths to reduce potential steric hindrance.

An anti-myostatin antibody can also be derivatized with a chemical groupsuch as polyethylene glycol (PEG), a methyl or ethyl group, or acarbohydrate group. These groups are useful to improve the biologicalcharacteristics of the antibody, e.g., to increase serum half-life.

Pharmaceutical Compositions and Kits

The invention also relates to compositions comprising a humananti-myostatin antibody with inhibitory properties.

The antagonist anti-myostatin antibodies of the invention are useful toprevent or treat a wide range of conditions and disorders in which it isdesirable to increase skeletal muscle mass and/or bone. In some cases,such conditions and disorders may be age related or disease related. Anantagonist anti-myostatin antibody of the invention may be used totreat, prevent or inhibit age-related loss of muscle mass and strengthincluding muscle atrophy that results from, e.g., immobilization.

In addition, an antagonist anti-myostatin antibody of the invention isuseful to treat or prevent muscle loss in wasting diseases or otherdiseases or conditions, associated with loss of muscle mass including,but not limited to, trauma (including muscle, nerve and bone trauma),burns, AIDS, cancer, hip fractures (especially in the elderly), jointreplacement, acute knee injuries, arthritis, chronic renal failure(CRF), congestive heart failure (CHF), chronic obstructive pulmonarydisease (COPD), multiple sclerosis (MS), Parkinson's Disease, chroniccritical illness, central nervous system (CNS) injury, stroke, cachexia,muscular dystrophies syndrome, surgery and joint injury.

Antagonist anti-myostatin antibodies of the invention also are useful totreat metabolic conditions. Such conditions include type 2 diabetesmellitus, metabolic syndromes such as syndrome X, insulin resistance,impaired glucose tolerance, and obesity.

Further, an antagonist anti-myostatin antibody of the invention isuseful to treat or prevent disorders associated with bone loss,including but not limited to age- or hormone-related osteoporosisosteopenia, osteoarthritis, and osteoporosis-related fractures.

Treatment may involve administration of one or more inhibitoryanti-myostatin monoclonal antibodies of the invention, orantigen-binding fragments thereof, alone or with a pharmaceuticallyacceptable carrier. Inhibitory anti-myostatin antibodies of theinvention and compositions comprising them, can be administered incombination with one or more other therapeutic, diagnostic orprophylactic agents. Additional therapeutic agents include anti-musculardystrophy agents (including steroids such as prednisone, deflazacort),and anabolic steroids, albuterol, nutritional supplements such ascreatine, and antibiotics such as gentamycin. Additional therapeuticagents also include anti-diabetes agents including but not limited tometformin, sulfonylureas, insulin, SYMLIN® (pramlintide), TZDs such asAVANDIA® (rosiglitazone) and ACTOS® (pioglitazone), GLP-1 analogs, suchexenitide, and DPP-IV inhibitors, such as LAF237. Further, additionaltherapeutic agents include anti-osteoporosis agents including but notlimit to bisphosphonates such as FOSAMAX® (alendronate) and ACTONEL®(risedronate), MIACALCIN® (calcitonin), EVISTA® (raloxifene), hormonalagents such estrogen and parathyroid. Such additional agents may beincluded in the same composition or administered separately. As usedherein, “pharmaceutically acceptable carrier” means any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Some examples of pharmaceutically acceptablecarriers are water, saline, phosphate buffered saline, dextrose,glycerol, ethanol and the like, as well as combinations thereof. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Additional examples of pharmaceutically acceptablesubstances are wetting agents or minor amounts of auxiliary substancessuch as wetting or emulsifying agents, preservatives or buffers, whichenhance the shelf life or effectiveness of the antibody.

The compositions of this invention may be in a variety of forms, forexample, liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions, tablets, pills, powders, liposomes and suppositories. Thepreferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans. The preferred mode ofadministration is parenteral (e.g., intravenous, subcutaneous,intraperitoneal, intramuscular). In a preferred embodiment, the antibodyis administered by intravenous infusion or injection. In anotherpreferred embodiment, the antibody is administered by intramuscular orsubcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the anti-myostatin antibodyin the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying that yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof. The proper fluidity of a solution canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

In one embodiment, the antibody is administered in a formulation as asterile aqueous solution having a pH that ranges from about 5.0 to about6.5 and comprising from about 1 mg/ml to about 200 mg/ml of antibody,from about 1 millimolar to about 100 millimolar of histidine buffer,from about 0.01 mg/ml to about 10 mg/ml of polysorbate 80, from about100 millimolar to about 400 millimolar of trehalose, and from about 0.01millimolar to about 1.0 millimolar of disodium EDTA dihydrate.

The antibodies of the present invention can be administered by a varietyof methods known in the art, although for many therapeutic applications,the preferred route/mode of administration is subcutaneous,intramuscular, or intravenous infusion. As will be appreciated by theskilled artisan, the route and/or mode of administration will varydepending upon the desired results.

In certain embodiments, the antibody compositions active compound may beprepared with a carrier that will protect the antibody against rapidrelease, such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems (J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978).

In certain embodiments, an anti-myostatin antibody of the invention canbe orally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) can also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the anti-myostatin antibodiescan be incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. To administer a compound of the inventionby other than parenteral administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to preventits inactivation.

Additional active compounds also can be incorporated into thecompositions. In certain embodiments, an inhibitory anti-myostatinantibody of the invention is co-formulated with and/or co-administeredwith one or more additional therapeutic agents. These agents include,without limitation, antibodies that bind other targets, antineoplasticagents, antitumor agents, chemotherapeutic agents, peptide analoguesthat inhibit myostatin, or antibodies or other molecules that bind toType II membrane receptors and prevent their binding to or activation bymyostatin. Such combination therapies may require lower dosages of theinhibitory anti-myostatin antibody as well as the co-administeredagents, thus avoiding possible toxicities or complications associatedwith the various monotherapies.

Inhibitory anti-myostatin antibodies of the invention and compositionscomprising them also may be administered in combination with othertherapeutic regimens, in particular in combination with exercise and/ordietary (including nutritional) supplements.

In certain embodiments, an inhibiting anti-myostatin antibody of theinvention is co-formulated with and/or co-administered with one or moreadditional therapeutic agents discussed, supra. These agents includethose that inhibit myostatin. Further, such combination therapies mayalso be used to treat, prevent or inhibit any of the aforementioneddiseases and conditions. Such combination therapies may require lowerdosages of the inhibitory anti-myostatin antibody as well as theco-administered agents, thus avoiding possible toxicities orcomplications associated with the various monotherapies.

The compositions of the invention may include a “therapeuticallyeffective amount” or a “prophylactically effective amount” of anantibody or antigen-binding portion of the invention. A “therapeuticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the antibody or antibody portion mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodyportion to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody or antibody portion are outweighedby the therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount may be less thanthe therapeutically effective amount.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus can be administered, several divided doses can be administeredover time or the dose can be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the anti-myostatin antibody or portion thereof andthe particular therapeutic or prophylactic effect to be achieved, and(b) the limitations inherent in the art of compounding such an antibodyfor the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe invention is 0.025 to 50 mg/kg, more preferably 0.1 to 50 mg/kg,more preferably 0.1-25, 0.1 to 10 or 0.1 to 3 mg/kg. In someembodiments, a formulation contains 5 mg/ml of antibody in a buffer of20 mM sodium citrate, pH 5.5, 140 mM NaCl, and 0.2 mg/ml polysorbate 80.It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

Another aspect of the present invention provides kits comprising ananti-myostatin antibody or antigen-binding portion of the invention or acomposition comprising such an antibody or portion. A kit may include,in addition to the antibody or composition, diagnostic or therapeuticagents. A kit can also include instructions for use in a diagnostic ortherapeutic method. In a preferred embodiment, the kit includes theantibody or a composition comprising it and a diagnostic agent that canbe used in a method described below. In another preferred embodiment,the kit includes the antibody or a composition comprising it and one ormore therapeutic agents that can be used in a method described below.

Diagnostic Methods of Use

In another aspect, the invention provides diagnostic methods. Theanti-myostatin antibodies can be used to detect myostatin in abiological sample in vitro or in vivo.

The anti-myostatin antibodies can be used in a conventional immunoassay,including, without limitation, an ELISA, an RIA, flow cytometry, tissueimmunohistochemistry, Western blot or immunoprecipitation. Theanti-myostatin antibodies of the invention can be used to detectmyostatin from humans. In another embodiment, the anti-myostatinantibodies can be used to detect myostatin from, for example, mice,cynomolgus monkeys, rat, dog, quail, chicken, turkey, pig and horse.

The invention provides a method for detecting myostatin in a biologicalsample comprising contacting the biological sample with ananti-myostatin antibody of the invention and detecting the boundantibody. In one embodiment, the anti-myostatin antibody is directlylabeled with a detectable label. In another embodiment, theanti-myostatin antibody (the first antibody) is unlabeled and a secondantibody or other molecule that can bind the anti-myostatin antibody islabeled. As is well known to one of skill in the art, a second antibodyis chosen that is able to specifically bind the particular species andclass of the first antibody. For example, if the anti-myostatin antibodyis a human IgG, then the secondary antibody could be an anti-human-IgG.Other molecules that can bind to antibodies include, without limitation,Protein A and Protein G, both of which are available commercially, e.g.,from Pierce Chemical Co.

Suitable labels for the antibody or secondary antibody have beendisclosed supra, and include various enzymes, prosthetic groups,fluorescent materials, luminescent materials and radioactive materials.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In other embodiments, myostatin can be assayed in a biological sample bya competition immunoassay utilizing myostatin standards labeled with adetectable substance and an unlabeled anti-myostatin antibody. In thisassay, the biological sample, the labeled myostatin standards and theanti-myostatin antibody are combined and the amount of labeled myostatinstandard bound to the unlabeled antibody is determined. The amount ofmyostatin in the biological sample is inversely proportional to theamount of labeled myostatin standard bound to the anti-myostatinantibody.

One can use the immunoassays disclosed above for a number of purposes.For example, the anti-myostatin antibodies can be used to detectmyostatin in cultured cells. In a preferred embodiment, theanti-myostatin antibodies are used to determine the amount of myostatinproduced by cells that have been treated with various compounds. Thismethod can be used to identify compounds that modulate myostatin proteinlevels. According to this method, one sample of cells is treated with atest compound for a period of time while another sample is leftuntreated. If the total level of myostatin is to be measured, the cellsare lysed and the total myostatin level is measured using one of theimmunoassays described above. The total level of myostatin in thetreated versus the untreated cells is compared to determine the effectof the test compound.

A preferred immunoassay for measuring total myostatin levels is flowcytometry or immunohistochemistry. Methods such as ELISA, RIA, flowcytometry, Western blot, immunohistochemistry, cell surface labeling ofintegral membrane proteins and immunoprecipitation are well known in theart. See, e.g., Harlow and Lane, supra. In addition, the immunoassayscan be scaled up for high throughput screening in order to test a largenumber of compounds for either activation or inhibition of myostatinexpression.

The anti-myostatin antibodies of the invention also can be used todetermine the levels of myostatin in a tissue or serum. In someembodiments, the tissue is a diseased tissue. In some embodiments of themethod, a tissue or a biopsy thereof is excised from a patient. Themethod comprises the steps of administering a detectably labeledanti-myostatin antibody or a composition comprising them to a patient inneed of such a diagnostic test and subjecting the patient to imaginganalysis to determine the location of the myostatin-expressing tissues.Imaging analysis is well known in the medical art, and includes, withoutlimitation, x-ray analysis, magnetic resonance imaging (MRI) or computedtomography (CT). The antibody can be labeled with any agent suitable forin vivo imaging, for example a contrast agent, such as barium, which canbe used for x-ray analysis, or a magnetic contrast agent, such as agadolinium chelate, which can be used for MRI or CT. Other labelingagents include, without limitation, radioisotopes, such as ⁹⁹Tc. Inanother embodiment, the anti-myostatin antibody will be unlabeled andwill be imaged by administering a second antibody or other molecule thatis detectable and that can bind the anti-myostatin antibody. Inembodiment, a biopsy is obtained from the patient to determine whetherthe tissue of interest expresses myostatin.

Therapeutic Methods of Use

In another embodiment, the invention provides a method for inhibitingmyostatin activity by administering an anti-myostatin antibody to apatient in need thereof. Any of the types of antibodies described hereinmay be used therapeutically. In various embodiments, the anti-myostatinantibody is a human, chimeric or humanized antibody. In a preferredembodiment, the myostatin is human and the patient is a human patient.Alternatively, the patient may be a mammal that expresses a myostatinthat the anti-myostatin antibody cross-reacts with. The antibody may beadministered to a non-human mammal expressing myostatin with which theantibody cross-reacts (i.e. a rat, a mouse, or a cynomolgus monkey) forveterinary purposes or as an animal model of human disease. Such animalmodels may be useful for evaluating the therapeutic efficacy ofantibodies of this invention.

These antibodies may be used in promoting muscle growth, weight gain andaiding in the prevention of frailty in livestock (cattle, swine, sheep,horses, chickens, turkeys and fish) and companion animals (dogs, catsand horses).

In another embodiment, the invention provides a method for promotingmuscle growth, weight gain and aiding in the prevention of frailty incattle, swine, sheep, chickens, turkeys, horses, fish, dogs and cats inneed thereof comprising the step of administering to said subject anantibody or antigen-binding portion as described herein.

As used herein, the term “a condition that may be prevented or treatedby reducing myostatin activity” is intended to include diseases andother disorders in which the presence of high levels of myostatin in asubject suffering from the disorder has been shown to be or is suspectedof being either responsible for the pathophysiology of the disorder or afactor that contributes to a worsening of the disorder. Such disordersmay be evidenced, for example, by an increase in the levels of myostatinin tissues and fluids or phosphorylated Smad 2 or 3 in muscle tissue inthe affected cells or tissues of a subject suffering from the disorder.The increase in myostatin levels may be detected, for example, using ananti-myostatin antibody as described above.

In another preferred embodiment, an anti-myostatin antibody may beadministered to a patient who expresses inappropriately high levels ofmyostatin. It is known in the art that high-level expression ofmyostatin can lead to a variety of wasting conditions, bone loss and toaccumulation of fat mass. In one embodiment, said method relates to thetreatment of diseases and conditions in which it is desirable todecrease the loss of or to increase skeletal muscle mass, to decreasebone loss or to increase bone mass and/or to reduce fat mass byneutralizing the effects of myostatin. Such conditions and diseases arementioned supra.

In another preferred embodiment, an anti-myostatin antibody may beadministered to a patient who does not express inappropriate levels ofmyostatin (high or low), but whose condition would still be successfullytreated or prevented by using anti-moystatin treatment. In oneembodiment, said method relates to the treatment of diseases andconditions in which it is desirable to inhibit moystatin activity totreat the loss of or to improve skeletal muscle mass, to treat bone lossor to increase bone mass and/or to treat fat mass by neutralizing theeffects of myostatin. Such diseases and conditions are known in the artand would include diseases and conditions that associate with a varietyof wasting conditions that can include bone loss and accumulation of fatmass, e.g., age-related degenerative diseases and non-degenerative,normal age-related conditions.

The antibody may be administered once, but more preferably isadministered multiple times. The antibody may be administered from threetimes daily to once every six months or longer. The administering may beon a schedule such as three times daily, twice daily, once daily, onceevery two days, once every three days, once weekly, once every twoweeks, once every month, once every two months, once every three monthsand once every six months. The antibody may also be administeredcontinuously via a minipump. The antibody may be administered via anoral, mucosal, buccal, intranasal, inhalable, intravenous, subcutaneous,intramuscular or parenteral route. The antibody may be administeredonce, at least twice or for at least the period of time until thecondition is treated, palliated or cured. The antibody generally will beadministered for as long as the condition is present. The antibody willgenerally be administered as part of a pharmaceutical composition asdescribed supra. The dosage of antibody will generally be in the rangeof 0.1-100 mg/kg, more preferably 0.5-50 mg/kg, more preferably 1-20mg/kg, and even more preferably 1-10 mg/kg. The serum concentration ofthe antibody may be measured by any method known in the art.

Co-administration of the antibody with an additional therapeutic agent(combination therapy) encompasses administering a pharmaceuticalcomposition comprising the anti-myostatin antibody and the additionaltherapeutic agent as well as administering two or more separatepharmaceutical compositions, one comprising the anti-myostatin antibodyand the other(s) comprising the additional therapeutic agent(s).Further, although co-administration or combination therapy generallymeans that the antibody and additional therapeutic agents areadministered at the same time as one another, it also encompassesinstances in which the antibody and additional therapeutic agents areadministered at different times. For instance, the antibody may beadministered once every three days, while the additional therapeuticagent is administered once daily. Alternatively, the antibody may beadministered prior to or subsequent to treatment of the disorder withthe additional therapeutic agent, for example after a patient has failedtherapy with the additional agent. Similarly, administration of theanti-myostatin antibody may be administered prior to or subsequent toother therapy, such as exercise and/or dietary (including nutritional)supplements. The antibody and one or more additional therapeutic agents(the combination therapy) may be administered once, twice or at leastthe period of time until the condition is treated, palliated or cured.Preferably, the combination therapy is administered multiple times. Thecombination therapy may be administered from three times daily to onceevery six months. The administering may be on a schedule such as threetimes daily, twice daily, once daily, once every two days, once everythree days, once weekly, once every two weeks, once every month, onceevery two months, once every three months and once every six months, ormay be administered continuously via a minipump. The combination therapymay be administered via an oral, mucosal, buccal, intranasal, inhalable,intravenous, subcutaneous, intramuscular or parenteral route.

Gene Therapy

The nucleic acid molecules of the present invention can be administeredto a patient in need thereof via gene therapy. The therapy may be eitherin vivo or ex vivo. In a preferred embodiment, nucleic acid moleculesencoding both a heavy chain and a light chain are administered to apatient. In a more preferred embodiment, the nucleic acid molecules areadministered such that they are stably integrated into chromosomes of Bcells because these cells are specialized for producing antibodies. In apreferred embodiment, precursor B cells are transfected or infected exvivo and re-transplanted into a patient in need thereof. In anotherembodiment, precursor B cells or other cells are infected in vivo usinga virus known to infect the cell type of interest. Typical vectors usedfor gene therapy include liposomes, plasmids and viral vectors.Exemplary viral vectors are retroviruses, adenoviruses andadeno-associated viruses. After infection either in vivo or ex vivo,levels of antibody expression can be monitored by taking a sample fromthe treated patient and using any immunoassay known in the art ordiscussed herein.

In a preferred embodiment, the gene therapy method comprises the stepsof administering an isolated nucleic acid molecule encoding the heavychain or an antigen-binding portion thereof of an anti-myostatinantibody and expressing the nucleic acid molecule. In anotherembodiment, the gene therapy method comprises the steps of administeringan isolated nucleic acid molecule encoding the light chain or anantigen-binding portion thereof of an anti-myostatin antibody andexpressing the nucleic acid molecule. In a more preferred method, thegene therapy method comprises the steps of administering of an isolatednucleic acid molecule encoding the heavy chain or an antigen-bindingportion thereof and an isolated nucleic acid molecule encoding the lightchain or the antigen-binding portion thereof of an anti-myostatinantibody of the invention and expressing the nucleic acid molecules. Thegene therapy method may also comprise the step of administering one ormore additional therapeutic agents, such as those mentioned, supra.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly and are not to be construed as limiting the scope of the inventionin any manner.

Example I Generation of Hybridomas Producing Anti-Myostatin Antibody

Antibodies of the invention were prepared, selected, and assayed asfollows: Eight to ten week old XENOMOUSE® mice were immunizedintraperitoneally or in their hind footpads with either a c-terminalmature myostatin (10 μg/dose/mouse) (R&D Systems, Catalog #788-G8) orwith a full-length cynomolgus mature myostatin. This dose was repeatedseven times over a three to four week period. Four days before fusion,the mice were given a final injection of the myostatin in PBS. Thespleen and lymph node lymphocytes from immunized mice were fused withthe non-secretory myeloma P3-X63-Ag8.653 cell line, and these fusedcells were subjected to HAT selection as previously described (Galfreand Milstein, Methods Enzymol. 73:3-46, 1981). A panel of hybridomas wasrecovered that all secrete myostatin specific human IgG2 or IgG4antibodies.

GDF8 and GDF11 ELISA Assay Protocol:

ELISA assay was used to detect antibody binding to myostatin or GDF11.Myostatin or GDF11 was coated onto a 96-well Immulon microtiter plate(NUNC-IMMUNO® plate MAXISORP® surface, Nalge Nunc International, Cat.No. 439454) at 4 μg/ml in 50 mM sodium bicarbonate buffer for overnightat 4° C. Plates were washed, and then blocked with phosphate-bufferedsaline (PBS) containing 0.1% Tween-20 and 0.5% bovine serum albumin.Antibodies were added to the blocked ELISA plates, incubated for 1 hour,and washed with PBS with Tween-20. Binding was detected by anti-humanIgG-horseradish peroxidase (Pierce Cat. No. 31420) followed by theaddition of ABTS (Pierce Cat. No. 37615). Colorimetric measurements wereperformed at 405 nm in a micro-plate reader (We used SpectraMax Plus384, Molecular Devices).

Eleven hybridomas were selected for further study and were designated1_(—)116_(—)1, 1_(—)136_(—)3, 1_(—)257_(—)1, 1_(—)46_(—)1, 2_(—)112_(—),1_(—)314_(—)1, 1_(—)66_(—)1, 2_(—)43_(—)1, 2_(—)177_(—)1, 1_(—)132_(—)1,and 1_(—)268_(—)1. The eleven hybridomas were deposited under terms inaccordance with the Budapest Treaty with the American Type CultureCollection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209 onFeb. 10, 2005. The hybridomas have been assigned the following referencenumbers:

Antibody Hybridoma Identifier ATCC Reference No. 1_116_1 PF8-1-116-1 (LN15902) PTA-6566 1_132_1 PF8-1-132-1 (LN 15903) PTA-6567 1_136_3PF8-1-136-3 (LN 15904) PTA-6568 1_257_1 PF8-1-257-1 (LN 15905) PTA-65691_268_1 PF8-1-268-1 (LN 15906) PTA-6570 1_314_1 PF8-1-314-1 (LN 15907)PTA-6571 1_46_1 PF8-1-46-1 (LN 15908) PTA-6572 1_66_1 PF8-1-66-1 (LN15909) PTA-6573 2_112_1 PF8-2-112-1 (LN 15910) PTA-6574 2_43_1PF8-2-43-1 (LN 15911) PTA-6575 2_177_1 PF8-2-177-1 (LN 15912) PTA-6576

Example II Sequences of Anti-Myostatin-Antibodies Prepared in Accordancewith the Invention

To analyze the structure of antibodies produced in accordance with theinvention, nucleic acids were cloned that encode heavy and light chainfragments from hybridomas producing anti-myostatin monoclonalantibodies.

Cloning and sequencing of the antibody variable regions, wasaccomplished as follows: Poly(A)⁺ mRNA was isolated using a Fast-Trackkit (Invitrogen) from approximately 2×10⁵ hybridoma cells derived fromXENOMOUSE® mice immunized with myostatin. cDNA was synthesized from themRNA by using random primers. The randomly primed cDNA was amplifiedusing human V_(H) or human VK family specific variable domain primers(Marks et al., “Oligonucleotide primers for polymerase chain reactionamplification of human immunoglobulin variable genes and design offamily-specific oligonucleotide probes.” Eur. J. Immunol. 21:985-991(1991)) or a universal human V_(H) primer [MG-30,5′-CAGGTGCAGCTGGAGCAGTCIGG-3] (SEQ ID NO: 91), in conjunction withprimers specific for the human Cγ₂ constant region, MG-40d[5′-GCTGAGGGAGTAGAGTCCTGAGGA-3′] (SEQ ID NO: 92) or a CK constant region[hKP2; as previously described in Green et al., 1994]. Nucleic acidmolecules were obtained that encode human heavy and kappa light chaintranscripts from the anti-myostatin producing hybridomas by PCRamplification from poly(A⁺) RNA using the primers described above. ThePCR products were cloned into [pCRII (Invitrogen)] using a TA cloningkit (Invitrogen) and both strands were sequenced using Prismdye-terminator sequencing kits (Applied Biosystems Inc) and an ABI 377sequencing machine (Applied Biosystems Inc). All sequences were analyzedby alignments to the “V BASE sequence directory” (Tomlinson et al., MRCCentre for Protein Engineering, Cambridge, UK) using MACVECTOR(Accelrys, San Diego, Calif.) and GENEWORKS (Hindmarsh, S. A. Australia)software programs.

To obtain the full-length expressed nucleic acid sequence of antibodiesproduced in accordance with the invention, nucleic acids were clonedthat encode full-length heavy and light chain coding regions fromhybridomas producing anti-myostatin antibodies. Cloning and sequencingwas accomplished as follows: Poly(A)⁺ mRNA was isolated using an RNeasyMini Kit (Qiagen) and cDNA synthesized from the mRNA with the AdvantageRT-for-PCR kit (BD Biosciences) using oligo(dT) priming. The oligo(dT)primed cDNA was amplified in two independent reactions using degenerateprimers designed to hybridize to the 5′ untranslated region (5′UTR) ofhuman V_(H) or human V_(κ) gene segments (TABLE 2A and 2B) inconjunction with non-degenerate primers specific for regions in the3′UTR of IGHG2 or IGHG4 [G_(—)3UTR_R, 5′TACGTGCCAAGCATCCTCGC] (SEQ IDNO: 93) or IGK [K_(—)3UTR_R, 5′AGGCTGGAACTGAGGAGCAGGTG] (SEQ ID NO: 94).Amplification was achieved using the Expand High Fidelity PCR kit(Roche) and a PTC-200 DNA Engine (MJ Research) with cycling as follows:2 minutes at 94° C.; 23× (30 seconds at 94° C., 50 seconds at 52° C., 2minutes at 72° C.); 8 minutes at 72° C. For both heavy and light chainreactions, the PCR products from two independent PCRs were cloned intopCR2.1 using a TOPO-TA cloning kit (Invitrogen) and both strands of twoclones were sequenced using Grills 16^(th) BDTv3.1/dGTP chemistry(Applied Biosystems Inc) and a 3730xl DNA Analyzer (Applied BiosystemsInc).

TABLE 2A Heavy and Light Chain 5' Amplification Primers: Primer NamePrimer Sequence SEQ ID NO VH1a_5UTR_F CCCTGAGAGCATCAYMYARMAACC 95VH3a_5UTR_F HVTHTCCACTYGGTGATCRGCACTG 96 VH3c_5UTR_FATTYRGTGATCAGSACTGAACASAG 97 VK1a_5UTR_F GSARTCAGWCYCWVYCAGGACACAGC 98VK2_5UTR_F CACCAGGKGATTTGCATATTRTCCC 99 VK3_5UTR_FATCAATGCCTGKGTCAGAGCYYTG 100

TABLE 2B 5′ Primers used for amplification of anti-myostatin antibodyheavy and light chains: Clone Forward Heavy Chain Primer Forward LightChain Primer 1_116_1 VH3a_5UTR_F VK1a_5UTR_F 1_132_1 VH1a_5UTR_FVK2_5UTR_F 1_136_3 VH3a_5UTR_F VK1a_5UTR_F 1_257_1 VH3a_5UTR_FVK1a_5UTR_F 1_314_1 VH3a_5UTR_F VK1a_5UTR_F 1_46_1 VH1a_5UTR_FVK2_5UTR_F 2_112_1 VH3c_5UTR_F VK3_5UTR_F 1_268_1 VH3a_5UTR_FVK1a_5UTR_F 1_66_1 VH3a_5UTR_F VK1a_5UTR_F 2_177_1 VH3c_5UTR_FVK3_5UTR_F 2_43_1 VH3c_5UTR_F VK3_5UTR_F

Gene Utilization Analysis

From the nucleic acid sequence and predicted amino acid sequence of theantibodies, the gene usage was identified for each antibody chain. Table3 sets forth the gene utilization of selected hybridoma clones ofantibodies in accordance with the invention:

TABLE 3 Heavy and Light Chain Gene Segment Utilization: Heavy Chain GeneUtilization Kappa Chain Gene Utilization Clone SEQ ID NO: V_(H) D_(H)J_(H) C_(H) SEQ ID NO: Vκ Jκ 1_116_1 1 V3-21 D5-5 JH4B G2 3 A30 JK11_132_1 5 V1-02 D4-23 JH6B G2 7 A3 JK4 1_136_3 9 V3-21 D5-5 JH4B G2 11A30 JK1 1_257_1 13 V3-21 D5-12 JH6B G2 15 A30 JK3 1_314_1 17 V3-21 —JH6B G2 19 A30 JK1 1_46_1 21 V1-02 D4-23 JH6B G2 23 A3 JK4 2_112_1 25V3-23 D1-7 JH3B G4 27 L2 JK4 1_268_1 29 V3-21 D1-26 JH4B G2 31 A30 JK41_66_1 33 V3-21 D5-5 JH4B G2 35 A30 JK3 2_177_1 37 V3-21 D1-7 JH3B G4 39L2 JK4 2_43_1 41 V3-23 D1-7 JH3B G4 43 L2 JK4

Mutations that developed during antibody maturation of the frameworkregions were modified back to the germline sequences as illustrated inthe sequence alignment of FIG. 19. Specifically, residue 12 in the heavychain of monoclonal antibody 2_(—)112_(—)1 (SEQ ID NOs: 26 and 77) wasmodified from Ile to Val. In the light chain (SEQ ID NOs:28 and 79),residue 58 was modified from Phe to Ile and residue 85 was modified fromIle to Val. Mutagenesis of specific residues of the heavy and lightchains of antibody 2_(—)112_(—)1 was carried out by designing primersand using the QuickChange Site Directed Mutagenesis Kit from Stratagene,according to the manufacturer's instructions. Mutations were confirmedby automated sequencing, and mutagenized inserts were subcloned intoexpression vectors. These expression vectors were transfected into NS0(ECACC # 85110503) and HEK-293T cells (American Type Culture Collection)to express recombinant antibodies of the invention. The resultingantibody having back-mutated framework regions is designated antibody2_(—)112_K.

Example III Inhibition of Myostatin by Human Anti-Myostatin Antibodies(A204 Luciferase Assay)

A myostatin responsive reporter gene assay (see, Thies, et al., GrowthFactors, (2001) 18:251-259.) was used to assess the biological activityof active myostatin in vitro. This assay uses a reporter vectorpGL3(CAGA)₁₅ coupled to luciferase. The CAGA box sequence (AGCCAGACA)(SEQ ID NO: 101) was reported to be a TGF-β-responsive element in thepromoter region of a TGF-β-induced gene, PAI-1. (Zawel, et al. Mol.Cell, (1998) 1:611-617). This reporter vector was generated by inserting15 copies of CAGA boxes into the SmaI and XhoI sites of pGL3-promotervector (Promega, Cat. No. E1761). The human rhabdomyosarcoma cell line,A204 (ATCC Cell No. HTB-82), was transiently transfected withpGL3(CAGA)15 and CMV β-GAL using Fugene 6 transfection reagent (RocheDiagnostics, Cat. No. 1814443). Transfected cells were cultured inMcCoy's 5A medium (Invitrogen, Cat. No. 16600) supplemented with 10%fetal bovine serum, 100 U/ml streptomycin, and 100 μg/ml penicillin for16 hours. Cells were changed to starvation medium (McCoy's 5A mediumwith streptomycin, penicillin, and 1 mg/ml bovine serum albumin), andthen treated with myostatin, GDF11, or activin for 6 hours at 37° C.Luciferase activity was measured using the DUAL-LIGHT® luciferase assaysystem (Applied Biosytems, Cat. No. T1004). Myostatin activates thereporter to about 10 fold with EC50 around 8 ng/ml. GDF11 activates thereporter in the very similar response. The neutralization activity of anantibody was determined by preincubating antibody with myostatin for 30minutes prior to addition to the A204 cells. As shown in FIG. 1, humananti-myostatin antibodies of the invention inhibitedmyostatin-stimulated luciferase activity in the A204 cells. A similarexperiment using GDF11 also was performed to study the specificities ofthe antibodies of the invention. The data is summarized in the FIG. 1.Most antibodies have much more potent neutralizing activity against GDF8than GDF11. Monoclonal antibodies 1_(—)46_(—)1 and 1_(—)132_(—)1neutralize both GDF8 and GDF11 with equal potency.

Example IV Inhibition of Myostatin by Human Anti-Myostatin Antibodies(L6 Beta-Lactamase Assay)

In vitro assays to measure myostatin binding to Type II membranereceptors in the presence of anti-myostatin antibodies were conducted todetermine if the anti-myostatin antibodies were capable of inhibitingsuch binding and their degree of inhibition.

An L6 Aurora reporter assay was performed to assess the biologicalactivity of active myostatin in vitro. L6 myostatin LD10 is a cell linestably expressing a beta-lactamase reporter gene. The promoter region inthe inserted reporter consists of 16 copies of Smad-binding element(SBE, GTCTAGAC (SEQ ID NO: 102), Zawel et al., Mol. Cell. 1: 611-17(1998)). The cells were cultured in DMEM high glucose (Invitrogen, Cat.No. 11995) supplemented with 10% heat-inactivated fetal bovine serum(FBS), 200-400 μg/ml Zeocin, 100 U/ml streptomycin, and 100 μg/mlpenicillin. Cells were plated to 96-well plate on day one and changed tothe starvation medium (DMEM with Zeocin, streptomycin, penicillin, and0.1% FBS) in afternoon of the second day. On the morning of the thirdday, cells were treated with myostatin, GDF11, or activin for 4 hours at37° C. Aurora CCF2 loading kit was used to analyze beta-lactamaseactivity according to the manufacture's suggestion (Invitrogen, Cat. No.K1032).

Myostatin activated smad-mediated expression of the reporter to about2-3 fold with EC50 around 5 ng/ml. GDF11 and activin activated thereporter in the very similar response.

The neutralization activity of an antibody was determined bypreincubating antibody with myostatin for 30 minutes prior to additionto the L6 myostatin LD10 cells. As shown in FIGS. 2 and 11, all humananti-myostatin antibodies tested inhibited smad protein activation in arange from about 1 to about 225 nM.

Example V Inhibition of Myostatin-Induced Smad 2 Phosphorylation(Western Blot)

HepG2 human hepatocellular carcinoma cells (ATTC Cell No. HB-8065) werecultured in DMEM high glucose (Invitrogen, Cat. No. 11995) supplementedwith 10% fetal bovine serum (FBS), 100 U/ml streptomycin, and 100 μg/mlpenicillin. 60-70% confluent cells were changed to the starvation medium(DMEM with streptomycin, penicillin, and 0.5% FBS) overnight. Cells weretreated with myostatin for 30 minutes. The neutralization activity of anantibody was determined by preincubating antibody with myostatin for 30minutes prior to addition to the HepG2 cells. Cells were lysed forwestern blot. Phosphorylated Smad2 was detected using a rabbitanti-phospho-Smad2 antibody (Cell Signalling, Cat. No. 3101), which wasthen detected by anti-Rabbit Alex 680 (Molecular Probes, Cat. No.A21076). The amount of phosphorylated Smad2 was quantified usingODYSSEY® Infrared Imager (L1-Cor). Glyseraldehyde-3-phosphatedehydrogenase (GAPDH) was used for normalization. As shown in FIG. 3,all of the neutralizing human anti-myostatin antibodies tested inhibitedsmad 2 phosphorylation.

Example VI Enhanced Expression of myf5 by Human Anti-MyostatinAntibodies

C2C12 myoblasts (ATCC Cell No. CRL-1772) were cultured in DMEM highglucose (Invitrogen, Cat. No. 11995) supplemented with 10% fetal bovineserum (FBS), 100 U/ml streptomycin, and 100 μg/ml penicillin. Cells weretreated with 300 ng/ml myostatin for 2 hours. The neutralizationactivity of an antibody was determined by preincubating antibody withmyostatin for 30 minutes prior to addition to the C2C12 cells. Cellswere harvested and RNA purified using RNeasy miniprep kit (Qiagen Cat.No. 74104). Purified RNA was quantified with RIBOGREEN® RNA QuantitationKit (Molecular Probes, Cat. No. R11490). Equal amount of total RNA wasused for real time PCR analysis to detect myf5 RNA expression.Primer/probe sets for mouse myf5 RNA detection were purchased fromASSAYS-ON-DEMAND® gene expression products (Applied Biosystems, Cat. No.Hs00271574_ml). Assay conditions used were according to themanufacture's recommendations. One-step RT-PCR was run on the sequencedetection system ABI7900 (Applied Biosystems). As shown in FIG. 4,neutralizing human anti-myostatin antibodies of the invention enhancedmyf5 expression. One non-neutralizing human anti-myostatin antibody,1_(—)159_(—)1, did not enhance myf5 expression.

Example VII Enhancement of Cell Differentiation by Human Anti-MyostatinAntibodies

The ability of antibodies to inhibit myostatin-mediated inhibition ofmyoblast differentiation was assessed in C2C12 myoblasts. C2C12 cellswere plated in 96-well plates at 25,000 cells/well in 200 μl DMEM/20%FBS/Penn-Strep. Twenty-four hours later, the media was aspirated, thecells rinsed 1× with HIT media (DMEM/2% horse serum/Penn-Strep), and 200μl HIT media alone or supplemented with 300 ng/ml GDF-8 (R&D Systems)alone or with various concentrations of antibody added. Forty-eighthours later, the media was aspirated and the cells lysed in 100 μl oflysis buffer (25 mM Tris pH 7.5, 3 mM EDTA, 1% NP-40, 1% Deoxycholate,0.1% SDS and 1 tablet of Roche Complete protease inhibitor/25 ml oflysis buffer). Embryonic myosin heavy chain (MHC) levels in the lysateswere determined by spotting 5 μl of lysate into individual wells of HighBind MA6000 plates (MSD#P11XB-1, Meso Scale Discovery, MSD), air dryingfor 1 hour, adding 100 μl of Blocker A (MSD#R93BA-2), incubating for 1hour at 25° C. with shaking, washing 4× with PBS/0.05% Tween-20, adding50 μl of embryonic MHC antibody (conditioned media from ATCC #CRL-2039hybridoma), incubating for 1.5 hours at 25° C. with shaking, washing 4×as before, adding 50 μl of Sulfo-TAG Anti-Mouse IgG (MSD #R32AC) whichhad been diluted 1:1000 in antibody diluent (MSD #R50AA-2), incubatingfor 1.5 hours at 25° C. with shaking, washing 4× as before, adding 150μl of 1× Read Buffer (MSD#R92TC-2) and analyzing chemiluminescence in aSector Imager 6000 (MSD). As shown in FIG. 5, neutralizing humananti-myostatin antibodies reversed myostatin-blocked muscledifferentiation in the MHC assay.

Example VIII Myostatin Peptide Binding

Eleven peptides were generated from mature GDF8 to test and localizeantibody binding (See FIG. 6). Peptide ELISA was done on glutaraldehydecoated ELISA plates and on uncoated ELISA plates (NUNC-IMMUNO® plateMAXISORP® surface, Nalge Nunc International, Cat. No. 439454).Glutaraldehyde coated plates were prepared by mixing 250 μlglutaraldehyde (50% w/v) with 50 ml deionized water and adding 200 μl ofthe solution into each well. Plates were incubated for at least 1 hourat room temperature. The glutaraldehyde was shaken out of the plate andsmashed upside down on paper towels to remove all the liquid prior touse.

ELISA plates were coated with peptide at 1.0 μg/well/50 μl (in Sodiumbicarbonate buffer, pH 9.6) for at least 4 hours, and then blocked with200 μl blocker (1% BSA, 1% Ethanolamine pH 7.4) for minimum 4 hours.Plates were washed with PBST three times with 200 μl/well. 50 μlantibody (1 μg/ml in PBST, Phosphate-buffered saline (PBS) with 0.05%Tween-20) sample was added to the wells for 1 hour. Plates were thenwashed with PBST three times. 50 μl anti-human IgG-horseradishperoxidase (HRP) (Pierce, Cat# 31420) diluted in PBST is added foranother hour. The wells were washed again with TBST three times, andthen with deionized water twice. 50 μl ABTS (MOSS) was added for 5-20minutes, and the signal was detected at 405 nm in a colormetric platereader. The results of the binding experiment are shown in FIG. 6. Theeleven peptides are shown in FIG. 7.

Example IX Epitope Mapping Studies

Cross-competition experiments were performed using the BIACORE® 3000instrument (Biacore International AB, Uppsala, Sweden and Piscataway,N.J.), following the manufacturer's protocols.

Experiments were performed in a BIACORE® 3000 instrument at 25° C. in0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Tween-20. Myostatinwas immobilized on a CM5 chip (Biacore™) using standard amine couplingprocedures yielding approximately 1300 RU (resonance units). Antibodysamples were prepared at 50 nM and injected in pairs, for example, areference mAb was injected for 10 minutes immediately followed by a 10minute injection of a test mAb. The surface was regenerated and theinjections were repeated in reverse order. The procedure was repeatedfor all possible pairs of antibodies. A cross-comparison of the totalresponse obtained for a pair of antibodies to that obtained forduplicate injections of each individual antibody was used to findantibodies that competitively bind with one another, e.g., theantibodies compete for binding to the antigen. A response greater thanthat observed for either of the duplicate injections was indicative ofbinding to different epitopes whereas a response intermediate to thoseobserved for the duplicate injections was indicative of binding to thesame or overlapping or adjacent epitopes. The results of this experimentare shown in FIG. 8 and summarized in FIG. 13.

Example X Immunoprecipitation of Myostatin from Cell Culture Medium

An immunoprecipitation study was conducted to test the binding ofmyostatin antibodies to mature GDF8 and mature GDF8/propeptide complex.293T cells were transfected with GDF-8 expression construct. Conditionedmedia was harvested 7 days after transfection, which contained matureGDF8 and latent complex. 100 μl conditioned media was incubated with 10μg myostatin antibodies for 16 hours at 4° C. Protein A Dynabeads(Dynal, Norway) were added and incubated for 90 minutes at 4° C. Theimmunoprecipitate was collected using MPC-E magnetic concentrator(Dynal, Norway), and washed four times with PBS. The immunoprecipitatewas resuspended in the reducing sample buffer and loaded on 4-12%Bis-Tris NuPage gel (Invitrogen). Western blotting was performed usingODYSSEY® infrared imaging system (L1-Cor). Mature GDF8 and unprocessedGDF8 were detected using goat anti-myostatin antibody (R&D systems, Cat#AF788). Rabbit anti-propeptide polyclonal antibody (made in-house) wasused to detect both propeptide and unprocessed GDF8. As shown in theFIG. 9, antibodies 2_(—)112_(—)1, 2_(—)43_(—)1, and 2_(—)177_(—)1immunoprecipitated mature GDF8, mature GDF8/propeptide complex, andunprocessed GDF8; antibody 1_(—)66_(—)1 immunoprecipitated mature GDF8and mature GDF8/propeptide complex. No other antibodiesimmunoprecipitated mature GDF8, propeptide, or unprocessed GDF8. Lane 4is the 1/10 medium loading control, and lane 7 is immunoprecipitationcontrol with medium only.

Example XI Immunoprecipitation of Myostatin from Mouse Serum

An immunoprecipitation study was conducted to test the binding ofmyostatin antibodies to mature GDF8 from mouse serum. 200 μl mouse serum(Rockland, Cat ## D208-00) was incubated with 20 μg myostatin antibodiesfor 16 hours at 4° C. Protein A Dynalbeads were added and incubated for90 minutes at 4° C. The immunoprecipitate was collected using MPC-Emagnetic concentrator (Dynal, Norway), and washed four times with PBS.The immunoprecipitate was resuspended in the reducing sample buffer andloaded on 4-12% Bis-Tris NuPage gel (Invitrogen). Western blotting wasperformed using ODYSSEY® Infrared imaging system (L1-Cor). Mature GDF8was detected using biotinylated goat anti-myostatin antibody (R&Dsystems, Cat# BAF788). As shown in the FIG. 10, antibodies2_(—)112_(—)1, 2_(—)43_(—)1, and 2_(—)177_(—)1 immunoprecipitated matureGDF8 from the mouse serum, whereas 1_(—)116_(—)1 and 1_(—)66_(—)1 couldnot. It is known that mature GDF8 in the serum binds to many inhibitoryproteins such as propeptide, FLRG, and GASAP1 (Hill et al. (2002) Themyostatin propeptide and the follistatin-related gene are inhibitorybinding proteins of myostatin in normal serum. J. Biol. Chem. 277:40735-40741. Hill et al. (2003) Regulation of myostatin in vivo bygrowth and differentiation factor-associated serum protein-1: a novelprotein with protease inhibitor and follistatin domains. Mol. Endocrin.17(6): 1144-1154). This experiment indicates that antibodies2_(—)112_(—)1, 2_(—)43_(—)1, and 2_(—)177_(—)1 are truly non-competitiveneutralizing antibodies, i.e., the antibodies neutralize myostatin butdo not block binding of inhibitory binding proteins present in serum.Such non-competitive neutralizing antibodies would be advantageous invivo.

Example XII Comparison of Anti-Myostatin Antibodies In Vivo

Five week old KKA^(y)/a mice (The Jackson Laboratory (Bar Harbor, Me.))were treated with 10 mg/kg of monoclonal antibodies 2_(—)43_(—)1,2_(—)177_(—)1, and 2_(—)112_K or vehicle (PBS) given subcutaneously onceper week for five weeks. At the end of the study animals were euthanizedand the gastrocnemius-plantaris-soleus (GPS), tibialis anterior (TA) andquadriceps (Quads) muscle groups were dissected and weighed. Treatmentwith the 2_(—)112_k antibody significantly increased (p<0.05) absolutemuscle mass for the GPS (+15.5%) and Quads (+30.6%). The 2_(—)177antibody significantly increased Quads (+11.5%) only. Although TAincreased following treatment with antibody 2_(—)112_K (+16.8%), theincrease was not statistically significant (p=0.330). Body weightincreased over the 5 weeks but was not significantly different betweentreatment groups at any time point during the study. When muscle masswas corrected for body weight (muscle mass index), there weresignificant differences only with 2_(—)112_K treatment (+11.4% in GPSand +20.6% in Quads). The results of this experiment are shown in FIGS.14 and 15.

Example XIII In Vivo Pharmacology and Efficacy of Antibody

The in vivo pharmacology and efficacy of antibody 2_(—)112_K wasevaluated in mice. To avoid potential immunomodulation due to a mouseanti-human antibody (MAHA) response, in vivo studies were performed inimmune-deficient Severe Combined Immunodecifient (SCID) beige mice.Subcutaneous administration of antibody 2_(—)112_K to these mice at 10mg/kg/week s.c. for 5 weeks increased skeletal muscle mass by 19-25% andmuscle strength by about 15%. Extending the duration of this treatmentto 10 weeks resulted in a further increase in muscle strength to 21% butno further increase in muscle mass (by 18-27%). Also, the Dose of 50mg/kg/week s.c. for 10 weeks did not yield superior increases in musclemass or strength compared with 10 mg/kg/week. The results of thisexperiment are shown in FIG. 16.

Example XIV Dose and Concentration Response Effects

When administered subcutaneously to SCID mice at doses ranging fromno-effect to maximum feasible dose (0.01, 0.1, 1 and 10 mg/kg/week),antibody 2_(—)112_K dose-dependently increased skeletal muscle mass byabout 0%, 3%, 7% and 28%, respectively, after 5 weeks (FIG. 17).Extending the duration of treatment to 10 weeks did not result in anyfurther increases in muscle mass but tended to increase tibialis musclestrength marginally by an additional 5%. Also, the antibodydose-dependently altered the body fat composition of these mice by +10%,+1%, −10%, −22% relative to PBS controls at the 0.01, 0.1, 1.0 and 10mg/kg/week for 5 weeks.

Non-linear, four-parameter analysis of the dose- andconcentration-response data yielded estimated ED50 and EC50 values of3.6 mg/kg/week and 20.4 mg/mL (−136 nM), respectively, assuming theincrease in muscle mass achieved with the 10 mg/kg/week dose to bemaximum efficacy. The relationship between the dose or serumconcentration of antibody 2_(—)112_K versus changes in muscle mass areshown in FIG. 18.

Example XV Efficacy of Antibody for Attenuation of Muscle Wasting

Ten week old KK mice (JAX laboratories, Bar Harbor, Me.) were randomizedinto three groups: placebo/vehicle, cortisone/vehicle, andcortisone/antibody 2_(—)112_K. Mice were dosed subcutaneously witheither vehicle (PBS) or 10 mg/kg antibody 2_(—)112_K for five weeksfollowed by implant of a placebo or cortisone pellet. Five days later,mice were euthanized, sacrificed and muscle mass and fat pad mass wereassessed. To assess muscle mass, the gastrocnemius-plantaris-soleus(GPS), tibialis anterior (TA) and quadriceps (quads) muscle groups weredissected and weighed. Cortisone implant resulted in a significantdecrease (p<0.05) in muscle mass to 85.1±2.2% (GPS), 85.5±3.9% (TA) and84.1±3.7% (quads) of the placebo/vehicle level. Treatment with antibody2_(—)112_K ameliorated entirely the cortisone-induced wasting ofplacebo/vehicle level for GPS ((102.0±2.2%), TA (99.5±3.6) and quads(104.6±2.8%). Values were similar when normalized per gram body weight.To test if this effect was specific for muscle mass, fat pad mass wasdetermined. For fat pad mass, inguinal, epididymal and abdominal fatpads were dissected and weighed. Treatment with antibody 2_(—)112-K didnot significantly (p<0.05) spare the loss in fat pad mass due tocortisone implant for inguinal, (cortisone/vehicle (75.1±4.7%) andcortisone/2_(—)112_K, (82.3±5.3%)), epididymal (cortisone/vehicle(79.3±5.0%) and cortisone/2_(—)112_K (87.1±3.9%)), or abdominal,(cortisone/vehicle (79.5±5.5%) and cortisone/2_(—)112_K (82.2±4.2%)),fat pads. Values are expressed relative to placebo/vehicle fat pad. Theresults of this experiment are shown in FIGS. 20A and 20B.

Example XVI Protein A Purification of Myostatin Antibody

1-2 liters of concentrated (10-fold) HEK293 media was filtered (2micron) and loaded on an equilibrated protein A column (AmershamBiosciences, Piscataway, N.J.) (53 mLs, equilibrated with 5 columnvolumes of D-PBS, pH 7.0) at 2 mL/min. The column was washed with 5column volumes of acetate buffer (20 mM sodium acetate, pH 5.5) and theprotein eluted with 4 column volumes of pH 3.2-3.5 sodium acetate (20mM). The pH of the eluate was adjusted to pH 5.5 with sodium hydroxideand filtered if needed. Finally the material was concentrated to 10mg/mL, dialyzed into 140 mM sodium chloride, 20 mM sodium acetate, pH5.5, filtered and stored at 4 degrees C.

All publications and patent applications cited in this specification areincorporated herein by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

1-23. (canceled)
 24. A method for promoting muscle growth in an animal,comprising the step of administering to said animal a monoclonalantibody or an antigen-binding portion thereof that specifically bindsto myostatin and inhibits myostatin activity, wherein said antibody orantigen-binding portion has at least one property selected from thegroup consisting of: (a) competes for binding to myostatin with anantibody selected from: 1_(—)116_(—)1 (PTA-6566); 1_(—)136_(—)3(PTA-6568); 1_(—)257_(—)1 (PTA-6569); 1_(—)46_(—)1 (PTA-6572);2_(—)112_(—)1 (PTA-6574); 1_(—)314_(—)1 (PTA-6571); 1_(—)66_(—)1(PTA-6573); 2_(—)43_(—)1 (PTA-6575); 2_(—)177_(—)1 (PTA-6576);1_(—)132_(—)1 (PTA-6567); or 1_(—)268_(—)1 (PTA-6570); (b) binds to thesame epitope of myostatin as an antibody selected from: 1_(—)116_(—)1(PTA-6566); 1_(—)136_(—)3 (PTA-6568); 1_(—)257_(—)1 (PTA-6569);1_(—)46_(—)1 (PTA-6572); 2_(—)112_(—)1 (PTA-6574); 1_(—)314_(—)1(PTA-6571); 1_(—)66_(—)1 (PTA-6573); 2_(—)43_(—)1 (PTA-6575);2_(—)177_(—)1 (PTA-6576); 1_(—)132_(—)1 (PTA-6567); or 1_(—)268_(—)1(PTA-6570); (c) binds to myostatin peptide 1 (SEQ ID NO: 103); (d) bindsto myostatin peptide 5 (SEQ ID NO:107); and (e) is cross-reactive withmyostatin peptide 1 (SEQ ID NO: 103) and myostatin peptide 5 (SEQ IDNO:107).
 25. The method of claim 24, wherein said antibody orantigen-binding portion selectively binds myostatin over Growth andDifferentiation Factor 11 (GDF11) by at least 50-fold.
 26. The method ofclaim 24, wherein said antibody or antigen-binding portion compriseshuman CDRs.
 27. The method of claim 24, wherein said antibody orantigen-binding portion is a non-competitive neutralizing antibody. 28.The method of claim 24, wherein said antibody or antigen-binding portionis chimeric.
 29. The method of claim 24, wherein the animal is a chickenor a turkey.
 30. The method of claim 24, wherein said antibody orantigen-binding portion comprises (a) the amino acid sequences of theheavy chain CDR1, CDR2, and CDR3 included in SEQ ID NO: 45 and the aminoacid sequences of the light chain CDR1, CDR2, and CDR3 included in SEQID NO: 47; (b) the amino acid sequences of the heavy chain CDR1, CDR2,and CDR3 included in SEQ ID NO: 49 and the amino acid sequences of thelight chain CDR1, CDR2, and CDR3 included in SEQ ID NO: 51; (c) theamino acid sequences of the heavy chain CDR1, CDR2, and CDR3 included inSEQ ID NO: 53 and the amino acid sequences of the light chain CDR1,CDR2, and CDR3 included in SEQ ID NO: 55; (d) the amino acid sequencesof the heavy chain CDR1, CDR2, and CDR3 included in SEQ ID NO: 57 andthe amino acid sequences of the light chain CDR1, CDR2, and CDR3included in SEQ ID NO: 59; (e) the amino acid sequences of the heavychain CDR1, CDR2, and CDR3 included in SEQ ID NO: 61 and the amino acidsequences of the light chain CDR1, CDR2, and CDR3 included in SEQ ID NO:63; (f) the amino acid sequences of the heavy chain CDR1, CDR2, and CDR3included in SEQ ID NO: 65 and the amino acid sequences of the lightchain CDR1, CDR2, and CDR3 included in SEQ ID NO: 67; (g) the amino acidsequences of the heavy chain CDR1, CDR2, and CDR3 included in SEQ ID NO:69 and the amino acid sequences of the light chain CDR1, CDR2, and CDR3included in SEQ ID NO: 71; (h) the amino acid sequences of the heavychain CDR1, CDR2, and CDR3 included in SEQ ID NO: 73 and the amino acidsequences of the light chain CDR1, CDR2, and CDR3 included in SEQ ID NO:75; (i) the amino acid sequences of the heavy chain CDR1, CDR2, and CDR3included in SEQ ID NO: 77 and the amino acid sequences of the lightchain CDR1, CDR2, and CDR3 included in SEQ ID NO: 79; (j) the amino acidsequences of the heavy chain CDR1, CDR2, and CDR3 included in SEQ ID NO:81 and the amino acid sequences of the light chain CDR1, CDR2, and CDR3included in SEQ ID NO: 83; (k) the amino acid sequences of the heavychain CDR1, CDR2, and CDR3 included in SEQ ID NO: 85 and the amino acidsequences of the light chain CDR1, CDR2, and CDR3 included in SEQ ID NO:87; or (l) the amino acid sequences of the heavy chain CDR1, CDR2, andCDR3 included in SEQ ID NO: 118 and the amino acid sequences of thelight chain CDR1, CDR2, and CDR3 included in SEQ ID NO: 120, whereinsaid CDR regions have less than 3 conservative amino acid substitutions.31. The method of claim 24, wherein: (a) the heavy chain of saidantibody or antigen-binding portion comprises the heavy chain CDR1, CDR2and CDR3 amino acid sequences of an antibody selected from:1_(—)116_(—)1 (PTA-6566); 1_(—)136_(—)3 (PTA-6568); 1_(—)257_(—)1(PTA-6569); 1_(—)46_(—)1 (PTA-6572); 2_(—)112_(—)1 (PTA-6574);1_(—)314_(—)1 (PTA-6571); 1_(—)66_(—)1 (PTA-6573); 2_(—)43_(—)1(PTA-6575); 2_(—)177_(—)1 (PTA-6576); 1_(—)132_(—)1 (PTA-6567); or1_(—)268_(—)1 (PTA-6570); (b) the light chain of said antibody orantigen-binding portion comprises the light chain CDR1, CDR2 and CDR3amino acid sequences of an antibody selected from: 1_(—)116_(—)1(PTA-6566); 1_(—)136_(—)3 (PTA-6568); 1_(—)257_(—)1 (PTA-6569);1_(—)46_(—)1 (PTA-6572); 2_(—)112_(—)1 (PTA-6574); 1_(—)314_(—)1(PTA-6571); 1_(—)66_(—)1 (PTA-6573); 2_(—)43_(—)1 (PTA-6575);2_(—)177_(—)1 (PTA-6576); 1_(—)132_(—)1 (PTA-6567); or 1_(—)268_(—)1(PTA-6570); or (c) said antibody or antigen-binding portion comprises aheavy chain of (a) and a light chain of (b).
 32. The method of claim 24,wherein said antibody or antigen-binding portion is selected from thegroup consisting of: (a) an antibody comprising the amino acid sequencesset forth in SEQ ID NO: 2 and SEQ ID NO:
 4. (b) an antibody comprisingthe amino acid sequences set forth in SEQ ID NO: 6 and SEQ ID NO: 8; (c)an antibody comprising the amino acid sequences set forth in SEQ ID NO:10 and SEQ ID NO: 12; (d) an antibody comprising the amino acidsequences set forth in SEQ ID NO: 14 and SEQ ID NO: 16; (e) an antibodycomprising the amino acid sequences set forth in SEQ ID NO: 18 and SEQID NO: 20; (f) an antibody comprising the amino acid sequences set forthin SEQ ID NO: 22 and SEQ ID NO: 24; (g) an antibody comprising the aminoacid sequences set forth in SEQ ID NO: 26 and SEQ ID NO: 28; (h) anantibody comprising the amino acid sequences set forth in SEQ ID NO: 30and SEQ ID NO: 32; (i) an antibody comprising the amino acid sequencesset forth in SEQ ID NO: 34 and SEQ ID NO: 36; (j) an antibody comprisingthe amino acid sequences set forth in SEQ ID NO: 38 and SEQ ID NO: 40;(k) an antibody comprising the amino acid sequences set forth in SEQ IDNO: 42 and SEQ ID NO: 44; and (l) an antibody comprising the amino acidsequences set forth in SEQ ID NO: 115 and SEQ ID NO:
 117. 33. The methodof claim 24, wherein said antibody or antigen-binding portion isselected from the group consisting of: (a) an antibody orantigen-binding portion comprising the variable domain amino acidsequences set forth in SEQ ID NO: 2 and SEQ ID NO:
 4. (b) an antibody orantigen-binding portion comprising the variable domain amino acidsequences set forth in SEQ ID NO: 6 and SEQ ID NO: 8; (c) an antibody orantigen-binding portion comprising the variable domain amino acidsequences set forth in SEQ ID NO: 10 and SEQ ID NO: 12; (d) an antibodyor antigen-binding portion comprising the variable domain amino acidsequences set forth in SEQ ID NO: 14 and SEQ ID NO: 16; (e) an antibodyor antigen-binding portion comprising the variable domain amino acidsequences set forth in SEQ ID NO: 18 and SEQ ID NO: 20; (f) an antibodyor antigen-binding portion comprising the variable domain amino acidsequences set forth in SEQ ID NO: 22 and SEQ ID NO: 24; (g) an antibodyor antigen-binding portion comprising the variable domain amino acidsequences set forth in SEQ ID NO: 26 and SEQ ID NO: 28; (h) an antibodyor antigen-binding portion comprising the variable domain amino acidsequences set forth in SEQ ID NO: 30 and SEQ ID NO: 32; (i) an antibodyor antigen-binding portion comprising the variable domain amino acidsequences set forth in SEQ ID NO: 34 and SEQ ID NO: 36; (j) an antibodyor antigen-binding portion comprising the variable domain amino acidsequences set forth in SEQ ID NO: 38 and SEQ ID NO: 40; (k) an antibodyor antigen-binding portion comprising the variable domain amino acidsequences set forth in SEQ ID NO: 42 and SEQ ID NO: 44; and (l) anantibody or antigen-binding portion comprising the variable domain aminoacid sequences set forth in SEQ ID NO: 115 and SEQ ID NO:
 117. 34. Themethod of claim 24, wherein said antibody or antigen-binding portioncomprises a first CDR sequence set comprising a first CDR1, first CDR2and first CDR3 and a second CDR sequence set comprising a second CDR1,second CDR2 and second CDR3, wherein said first CDR set and said secondCDR set each sequentially together have at least 90% identity to theCDR1, CDR2 and CDR3 sequences, sequentially together, of: (a) SEQ ID NO:2 and SEQ ID NO: 4, respectively; (b) SEQ ID NO: 6 and SEQ ID NO: 8,respectively; (c) SEQ ID NO:10 and SEQ ID NO:12, respectively; (d) SEQID NO:14 and SEQ ID NO:16, respectively; (e) SEQ ID NO:18 and SEQ IDNO:20, respectively; (f) SEQ ID NO:22 and SEQ ID NO:24, respectively;(g) SEQ ID NO:26 and SEQ ID NO:28, respectively; (h) SEQ ID NO:30 andSEQ ID NO:32, respectively; (i) SEQ ID NO:34 and SEQ ID NO:36,respectively; (j) SEQ ID NO:38 and SEQ ID NO:40, respectively; (k) SEQID NO:42 and SEQ ID NO:44, respectively; and (l) SEQ ID NO:115 and SEQID NO:117, respectively.
 35. The method of claim 24, wherein saidantibody or antigen-binding portion comprises a first CDR sequence setcomprising a first CDR1, first CDR2 and first CDR3 and a second CDRsequence set comprising a second CDR1, second CDR2 and second CDR3,wherein said first CDR set and said second CDR set are each the CDR1,CDR2 and CDR3 sequences, sequentially together, of: (a) SEQ ID NO: 2 andSEQ ID NO: 4, respectively; (b) SEQ ID NO: 6 and SEQ ID NO: 8,respectively; (c) SEQ ID NO:10 and SEQ ID NO:12, respectively; (d) SEQID NO:14 and SEQ ID NO:16, respectively; (e) SEQ ID NO:18 and SEQ IDNO:20, respectively; (f) SEQ ID NO:22 and SEQ ID NO:24, respectively;(g) SEQ ID NO:26 and SEQ ID NO:28, respectively; (h) SEQ ID NO:30 andSEQ ID NO:32, respectively; (i) SEQ ID NO:34 and SEQ ID NO:36,respectively; (j) SEQ ID NO:38 and SEQ ID NO:40, respectively; (k) SEQID NO:42 and SEQ ID NO:44, respectively; and (l) SEQ ID NO:115 and SEQID NO:117, respectively.
 36. The method of claim 24, wherein saidantibody is produced by a cell having ATCC Deposit Designation Numberselected from the group consisting of PTA-6566, PTA-6567, PTA-6568,PTA-6569, PTA-6570, PTA-6571, PTA-6572, PTA-6573, PTA-6574, PTA-6575,and PTA-6576.
 37. The method of claim 24, wherein said monoclonalantibody or antigen-binding portion binds to myostatin peptide 1 (SEQ IDNO: 103).
 38. The method of claim 24, wherein said monoclonal antibodyor antigen-binding portion binds to myostatin peptide 5 (SEQ ID NO:107).39. The method of claim 24, wherein said monoclonal antibody orantigen-binding portion is cross-reactive with myostatin peptide 1 (SEQID NO: 103) and myostatin peptide 5 (SEQ ID NO:107).
 40. The method ofclaim 24, wherein said antibody or antigen-binding portion competes forbinding to myostatin with an antibody selected from: 1_(—)116_(—)1(PTA-6566); 1_(—)136_(—)3 (PTA-6568); 1_(—)257_(—)1 (PTA-6569);1_(—)46_(—)1 (PTA-6572); 2_(—)112_(—)1 (PTA-6574); 1_(—)314_(—)1(PTA-6571); 1_(—)66_(—)1 (PTA-6573); 2_(—)43_(—)1 (PTA-6575);2_(—)177_(—)1 (PTA-6576); 1_(—)132_(—)1 (PTA-6567); or 1_(—)268_(—)1(PTA-6570).
 41. The method of claim 24, wherein said antibody orantigen-binding portion binds to the same epitope of myostatin as anantibody selected from: 1_(—)116_(—)1 (PTA-6566); 1_(—)136_(—)3(PTA-6568); 1_(—)257_(—)1 (PTA-6569); 1_(—)46_(—)1 (PTA-6572);2_(—)112_(—)1 (PTA-6574); 1_(—)314_(—)1 (PTA-6571); 1_(—)66_(—)1(PTA-6573); 2_(—)43_(—)1 (PTA-6575); 2_(—)177_(—)1_(PTA-6576);1_(—)132_(—)1 (PTA-6567); or 1_(—)268_(—)1 (PTA-6570).